Loci associated charcoal rot drought complex tolerance in soybean

ABSTRACT

The invention relates to methods and compositions for identifying soybean plants that are tolerant, have improved tolerance or are susceptible to Charcoal Rot Drought Complex. The methods use molecular genetic markers to identify, select and/or construct tolerant plants or identify and counter-select susceptible plants. Soybean plants that display tolerance or improved tolerance to Charcoal Rot Drought Complex that are generated by the methods of the invention are also a feature of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/872,927filed Apr. 29, 2013 which is pending, which is a divisional of U.S.application Ser. No. 12/342,218 filed Dec. 23, 2008, now U.S. Pat. No.8,329,982 granted Dec. 11, 2012, and further claims the benefit of U.S.application Ser. No. 12/342,189 filed Dec. 23, 2008, now U.S. Pat. No.8,212,108 granted Jul. 3, 2012, U.S. application Ser. No. 12/342,204filed Dec. 23, 2008, now U.S. Pat. No. 8,450,558 granted May 20, 2013,U.S. Application Ser. No. 61/082,024 filed Jul. 18, 2008, and U.S.Application Ser. No. 61/009,643 filed Dec. 31, 2007, all of which areeach herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods foridentifying soybean plants that are tolerant, have improved tolerance,or are susceptible to Charcoal Rot Drought Complex, where the methodsuse molecular genetic markers to identify, select and/or constructdisease and/or drought-tolerant plants. The invention also relates tosoybean plants that display tolerance or improved tolerance to CharcoalRot Drought Complex that are generated by the methods of the invention.

BACKGROUND OF THE INVENTION

Soybean, a legume, has become the world's primary source of seed oil andseed protein. In addition, its utilization is being expanded to theindustrial, manufacturing and pharmaceutical sectors. Soybeanproductivity is a vital agricultural and economic consideration.Unfortunately, soybean is host to one of the widest ranges of infectiouspathogens of all crops. More than a hundred different pathogens areknown to affect soybean plants, some of which pose significant economicthreats. Improving soybean disease tolerance to these many pathogens iscrucial to preventing yield losses.

Charcoal Rot and Drought

Charcoal rot is caused by the fungus Macrophomina phaseolina. The fungushas a particularly wide geographic distribution and is found throughoutthe world. M. phaseolina is most severe between 35° North and 35° Southlatitude (Wyllie (1976) ‘Macrophomina phaseolina—Charcoal Rot’ P482-484In L. D. Hill (ed.) World Soybean Research Proc of the World SoybeanRes. Conf., Champaign, Ill. Interstate, Danville, Ill.). The fungus alsohas a wide host range and infects over 500 crop and weed species and ishighly variable. Known major crop hosts include alfalfa, maize, cotton,grain sorghum, peanut and soybean.

Symptoms of charcoal rot on soybean can appear during any growth stage.Infected seeds germinate, but usually die within a few days. The fungusalso invades seedlings and may or may not exhibit symptoms, but servesas latent sources of inoculum for later in the growing season. The mostcommon charcoal rot symptoms appear later in the season. Initially,disease plants exhibit smaller leaflet size, reduced height, andwilting. Ultimately, M. phaseolina can reduce plant height, root volume,and root weight by more than 50%. These deleterious effects on roots aremost evident during the pod formation and seed filling stages, whendemand for water is high. Affected plants mature several weeks earlierthan normal and seed weight, number, and quality are reduced (Smith &Wyllie (1999) ‘Charcoal rot’ In G. L. Hartman (ed.) Compendium ofsoybean diseases. 4^(th) ed. APS Press, St. Paul, Minn.).

High ambient temperatures and low water availability exacerbate charcoalrot symptoms in soybean. Thus, charcoal rot is primarily known as a dryweather or drought induced disease. Symptoms caused by M. phaseolina areoften attributed drought stress.

In localized areas, yield losses can be as high as 90%. In the periodfrom 1996-2005, charcoal rot was the third leading cause of soybeanyield loss in the U.S. Average annual losses were 29MM bushels resultingin approximately $188MM annual income loss. Only soybean cyst nematodeand phythophthora root rot caused greater economic loss during thatperiod (Wrather & Koenning (2006) ‘Soybean Disease Loss Estimates forthe United States, 1996-2006’. University of Missouri—ColumbiaAgriculture Experiment Station. November 2006 published on the internetat aes.missouri.edu/delta/research/soyloss.stm Dec. 5, 2007).

Complete or vertical resistance to M. phaseolina has not been identifiedin soybean, which strongly suggests that a single gene conferringresistance does not exist. In most field and greenhouse evaluations, thegreat majority of soybean cultivars have been found to be either highlyor moderately susceptible to M. phaseolina. Only a few cultivars havebeen identified as possessing partial or horizontal resistance (Smith &Carville (1997) ‘Field screening of commercial and experimental soybeancultivars for their reaction to Macrophomina phaseolina’ Plant Dis81:804-809).

An alternative approach to identifying complete resistance is toidentify plants that show phenotypic tolerance to a particular pathogen.Tolerance can be described as the relative ability of a plant to surviveinfection without showing severe symptoms such as death, stunting, lossof vigor or yield loss. Tolerance includes any mechanism other thanwhole-plant immunity or resistance that reduces the expression ofsymptoms indicative of infection. Infected plants that exhibit tolerancewill yield nearly as well as uninfected plants. However, phenotypicselection requires pathogenic infection which has many advantages.

The development of molecular genetic markers has facilitated mapping andselection of agriculturally important traits in soybean. Markers tightlylinked to disease tolerance genes are an asset in the rapididentification of tolerant soybean lines on the basis of genotype by theuse of marker assisted selection (MAS). Introgressing disease tolerancegenes into a desired cultivar would also be facilitated by usingsuitable DNA markers.

Molecular Markers and Marker Assisted Selection

A genetic map is a graphical representation of a genome (or a portion ofa genome such as a single chromosome) where the distances betweenlandmarks on the chromosome are measured by the recombinationfrequencies between the landmarks. A genetic landmark can be any of avariety of known polymorphic markers, for example but not limited to,molecular markers such as SSR markers, RFLP markers, or SNP markers.Furthermore, SSR markers can be derived from genomic or expressednucleic acids (e.g., ESTs). The nature of these physical landmarks andthe methods used to detect them vary, but all of these markers arephysically distinguishable from each other (as well as from theplurality of alleles of any one particular marker) on the basis ofpolynucleotide length and/or sequence.

Although specific DNA sequences which encode proteins are generallywell-conserved across a species, other regions of DNA (typicallynon-coding) tend to accumulate polymorphism, and therefore, can bevariable between individuals of the same species. Such regions providethe basis for numerous molecular genetic markers.

In general, any differentially inherited polymorphic trait (includingnucleic acid polymorphism) that segregates among progeny is a potentialmarker. The genomic variability can be of any origin, for example,insertions, deletions, duplications, repetitive elements, pointmutations, recombination events or the presence and sequence oftransposable elements. A large number of soybean molecular markers areknown in the art, and are published or available from various sources,such as the SOYBASE internet resource. Similarly, numerous methods fordetecting molecular markers are also well-established.

The primary motivation for developing molecular marker technologies fromthe point of view of plant breeders has been the possibility to increasebreeding efficiency through marker assisted selection (MAS). A molecularmarker allele that demonstrates linkage disequilibrium with a desiredphenotypic trait (e.g., a quantitative trait locus, or QTL, such asresistance to a particular disease) provides a useful tool for theselection of a desired trait in a plant population. The key componentsto the implementation of this approach are: (i) the creation of a densegenetic map of molecular markers, (ii) the detection of QTL based onstatistical associations between marker and phenotypic variability,(iii) the definition of a set of desirable marker alleles based on theresults of the QTL analysis, and (iv) the use and/or extrapolation ofthis information to the current set of breeding germplasm to enablemarker-based selection decisions to be made.

The availability of integrated linkage maps of the soybean genomecontaining increasing densities of public soybean markers hasfacilitated soybean genetic mapping and MAS. See, e.g., Cregan, et al.,(1999) “An Integrated Genetic Linkage Map of the Soybean Genome” CropSci 39:1464-1490; Song, et al., (2004) “A New Integrated Genetic LinkageMap of the Soybean” Theor Appl Genet 109:122-128; Diwan & Cregan (1997)“Automated sizing of fluorescent-labeled simple sequence repeat (SSR)markers to assay genetic variation in Soybean” Theor Appl Genet95:220-225; the SOYBASE resources on the world wide web, including theShoemaker Lab Home Page and other resources that can be accessed throughSOYBASE; and see, the Soybean Genomics and Improvements Laboratory(SGIL) website on the world wide web, and see especially the Cregan Labwebpage.

Two types of markers are frequently used in marker assisted selectionprotocols, namely simple sequence repeat (SSR, also known asmicrosatellite) markers, and single nucleotide polymorphism (SNP)markers. The term SSR refers generally to any type of molecularheterogeneity that results in length variability, and most typically isa short (up to several hundred base pairs) segment of DNA that consistsof multiple tandem repeats of a two or three base-pair sequence. Theserepeated sequences result in highly polymorphic DNA regions of variablelength due to poor replication fidelity, e.g., caused by polymeraseslippage. SSRs appear to be randomly dispersed through the genome andare generally flanked by conserved regions. SSR markers can also bederived from RNA sequences (in the form of a cDNA, a partial cDNA or anEST) as well as genomic material.

The characteristics of SSR heterogeneity make them well suited for useas molecular genetic markers; namely, SSR genomic variability isinherited, is multiallelic, codominant and is reproducibly detectable.The proliferation of increasingly sophisticated amplification-baseddetection techniques (e.g., PCR-based) provides a variety of sensitivemethods for the detection of nucleotide sequence heterogeneity. Primers(or other types of probes) are designed to hybridize to conservedregions that flank the SSR domain, resulting in the amplification of thevariable SSR region. The different sized amplicons generated from an SSRregion have characteristic and reproducible sizes. The different sizedSSR amplicons observed from two homologous chromosomes in an individual,or from different individuals in the plant population are generallytermed “marker alleles.” As long as there exists at least two SSRalleles that produce PCR products with at least two different sizes, theSSRs can be employed as a marker.

Soybean markers that rely on single nucleotide polymorphisms (SNPs) arealso well known in the art. Various techniques have been developed forthe detection of SNPs, including allele specific hybridization (ASH;see, e.g., Coryell, et al. (1999) “Allele specific hybridization markersfor soybean,” Theor Appl Genet 98:690-696). Additional types ofmolecular markers are also widely used, including but not limited toexpressed sequence tags (ESTs) and SSR markers derived from ESTsequences, restriction fragment length polymorphism (RFLP), amplifiedfragment length polymorphism (AFLP), randomly amplified polymorphic DNA(RAPD) and isozyme markers. A wide range of protocols are known to oneof skill in the art for detecting this variability, and these protocolsare frequently specific for the type of polymorphism they are designedto detect. For example, PCR amplification, single-strand conformationpolymorphisms (SSCP) and self-sustained sequence replication (3SR; see,Chan & Fox, (1999) “NASBA and other transcription-based amplificationmethods for research and diagnostic microbiology,” Rev Med Microbiol10:185-196).

Linkage of one molecular marker to another molecular marker is measuredas a recombination frequency. In general, the closer two loci (e.g., twoSSR markers) are on the genetic map, the closer they lie to each otheron the physical map. A relative genetic distance (determined by crossingover frequencies, measured in centimorgans; cM) is generallyproportional to the physical distance (measured in base pairs, e.g.,kilobase pairs [kb] or megabase pairs [Mbp]) that two linked loci areseparated from each other on a chromosome. A lack of preciseproportionality between cM and physical distance can result fromvariation in recombination frequencies for different chromosomalregions, e.g., some chromosomal regions are recombinational “hot spots,”while others regions do not show any recombination, or only demonstraterare recombination events. In general, the closer one marker is toanother marker, whether measured in terms of recombination or physicaldistance, the more strongly they are linked. In some aspects, the closera molecular marker is to a gene that encodes a polypeptide that impartsa particular phenotype (disease tolerance), whether measured in terms ofrecombination or physical distance, the better that marker serves to tagthe desired phenotypic trait.

Genetic mapping variability can also be observed between differentpopulations of the same crop species, including soybean. In spite ofthis variability in the genetic map that may occur between populations,genetic map and marker information derived from one population generallyremains useful across multiple populations in identification of plantswith desired traits, counter-selection of plants with undesirable traitsand in guiding MAS.

QTL Mapping

It is the goal of the plant breeder to select plants and enrich theplant population for individuals that have desired traits, for example,pathogen tolerance, leading ultimately to increased agriculturalproductivity. It has been recognized for quite some time that specificchromosomal loci (or intervals) can be mapped in an organism's genomethat correlate with particular quantitative phenotypes. Such loci aretermed quantitative trait loci, or QTL. The plant breeder canadvantageously use molecular markers to identify desired individuals byidentifying marker alleles that show a statistically significantprobability of co-segregation with a desired phenotype (e.g., pathogenicinfection tolerance), manifested as linkage disequilibrium. Byidentifying a molecular marker or clusters of molecular markers thatco-segregate with a quantitative trait, the breeder is thus identifyinga QTL. By identifying and selecting a marker allele (or desired allelesfrom multiple markers) that associates with the desired phenotype, theplant breeder is able to rapidly select a desired phenotype by selectingfor the proper molecular marker allele (a process called marker-assistedselection, or MAS). The more molecular markers that are placed on thegenetic map, the more potentially useful that map becomes for conductingMAS.

Multiple experimental paradigms have been developed to identify andanalyze QTL (see, e.g., Jansen, (1996) Trends Plant Sci 1:89). In thisstudy we utilized “Intergroup Allele Frequency Distribution” analysisusing GeneFlow™ version 7.0 software. An intergroup allele frequencydistribution analysis provides a method for finding non-randomdistributions of alleles between two phenotypic groups.

During processing, a contingency table of allele frequencies isconstructed and from this a G-statistic and probability are calculated(the G statistic is adjusted by using the William's correction factor).The probability value is adjusted to take into account the fact thatmultiple tests are being done (thus, there is some expected rate offalse positives). The adjusted probability is proportional to theprobability that the observed allele distribution differences betweenthe two classes would occur by chance alone. The lower that probabilityvalue, the greater the likelihood that the Charcoal Rot infectionphenotype and the marker will co-segregate. A more complete discussionof the derivation of the probability values can be found in theGeneFlow™ version 7.0 software documentation. See, also, Sokal & Rolf,(1981), Biometry: The Principles and Practices of Statistics inBiological Research, 2nd ed., San Francisco, W. H. Freeman and Co.

The underlying logic is that markers with significantly different alleledistributions between the tolerant and susceptible groups (i.e., nonrandom distributions) might be associated with the trait and can be usedto separate them for purposes of marker assisted selection of soybeanlines with previously uncharacterized tolerance or susceptibility. Thepresent analysis examined one marker locus at a time and determined ifthe allele distribution within the tolerant group is significantlydifferent from the allele distribution within the susceptible group. Astatistically different allele distribution is an indication that themarker is linked to a locus that is associated with reaction to thetrait of interest. In this analysis, unadjusted probabilities less thanone are considered significant (the marker and the phenotype showlinkage disequilibrium), and adjusted probabilities less thanapproximately 0.05 are considered highly significant. Allele classesrepresented by less than 5 observations across both groups were notincluded in the statistical analysis. In addition, in this study weutilized “Trait Allele Frequency Analysis” using GeneFlow™ version 7.0software. For the Trait Allele Correlation report you must selectaccessions, markers and a single trait. For each allele at each selectedmarker, the report will show you the effect of having 0, 1 or 2 doses ofthat allele on the trait of interest. For each dosage comparison itcalculates a t-statistic, probability and adjusted probability bycomparing the means of two different dosage classes. The adjustedprobability gives you a better idea of the experiment-wise significancegiven the number of alleles being tested, and is calculated asP_adj=(1−((1−Prob)**n)) where n is the number of tests being done inthis analysis (see, Experimental Design: Procedures for the BehavioralSciences). A more complete discussion of the derivation of theprobability values can be found in the GeneFlow version 7.0 softwaredocumentation. See also, Sokal & Rolf, (1995) Biometry, 3rd ed., SanFrancisco, W.H. Freeman and Co.

There is a need in the art for improved soybean strains that aretolerant to Charcoal Rot and its causative agents, namely Macrophominaphaseolina infection and low-available water growth conditions. There isa need in the art for methods that identify soybean plants orpopulations (germplasm) that display tolerance to Charcoal Rot DroughtComplex. What is needed in the art is to identify molecular geneticmarkers that are linked to Charcoal Rot Drought Complex tolerance lociin order to facilitate MAS. Such markers can be used to selectindividual plants and plant populations that show favorable markeralleles in soybean populations and then employed to select the tolerantphenotype, or alternatively, be used to counterselect plants or plantpopulations that show a Charcoal Rot Drought Complex susceptibilityphenotype. The present invention provides these and other advantages.

SUMMARY OF THE INVENTION

Compositions and methods for identifying soybean plants or germplasmwith tolerance to Charcoal Rot Drought Complex are provided. Methods ofmaking soybean plants or germplasm that are tolerant to Charcoal RotDrought Complex, e.g., through introgression of desired tolerance markeralleles and/or by transgenic production methods, as well as plants andgermplasm made by these methods, are also provided. Systems and kits forselecting tolerant plants and germplasm are also a feature of theinvention.

Charcoal Rot is a major disease of soybean, causing severe losses insoybean viability and overall yield. Charcoal Rot is caused by infectionof the plant with the pathogenic fungus Macrophomina phaseolina. WhileCharcoal Rot is more prevalent during periods of low-available watergrowth conditions, making such conditions another causative factor ofCharcoal Rot, Charcoal Rot can exist in the absence of such growthconditions. Macrophomina resistant soybean cultivars have been producedin an attempt to reduce losses due to Charcoal Rot. However, the strongselective pressures that resistant soybean impose on Macrophomina arelikely to cause relatively rapid loss of the resistance phenotype, ashas been seen with other fungal pathogens of soybean, such asSclerotinia. In contrast, tolerance to Charcoal Rot or its causativeagents, Macrophomina infection and/or low-available water growthconditions, in which the plant survives and produces high yields,despite a productive Macrophomina infection, is an alternate strategy tocombat losses due to Charcoal Rot. Such tolerance provides advantagesover pathogen resistance. Selection for tolerance in the plant is lesslikely to result in the evolution of destructive races of Macrophominathat combat and overcome the tolerance traits, leading to ahost/pathogen relationship that more resembles commensalism as opposedto parasitism.

Further, low-available water growth conditions, e.g. drought, is and hasalways been a major cause of soybean damage, causing severe losses insoybean viability and overall yield. Because of this, soybean plantstolerant to low-available water growth conditions are desirable outsideof the realm of Charcoal Rot tolerance; tolerance to low-available watergrowth conditions would have economic benefits even in the absence ofCharcoal Rot. Therefore, tolerance to low-available water growthconditions is a desirable trait in soybeans both alone and for itseffects on Charcoal Rot tolerance.

The identification and selection of soybean plants that show toleranceto Charcoal Rot Drought Complex using MAS can provide an effective andenvironmentally friendly approach to overcoming losses caused by theseconditions. The present invention provides a number of soybean markerloci and QTL chromosome intervals that demonstrate statisticallysignificant co-segregation with Charcoal Rot Drought Complex tolerance.Detection of these QTL markers or additional loci linked to the QTLmarkers can be used in marker-assisted soybean breeding programs toproduce tolerant plants or plants with improved tolerance.

In some aspects, the invention provides methods for identifying a firstsoybean plant or germplasm (e.g., a line or variety) that has tolerance,improved tolerance, or susceptibility to Charcoal Rot Drought Complex.In the methods, at least one allele of one or more marker locus (e.g., aplurality of marker loci) that is associated with the tolerance,improved tolerance, or susceptibility is detected in the first soybeanplant or germplasm. The marker loci can be selected from the lociprovided in FIG. 1, including Sct_028, Satt512, S60211-TB, Sat_117,S01954-1-A, P13158A, S63880-CB, S00415-1-A, S00705-1-A, and S02118-1-A,as well as any other marker that is linked to these QTL markers (e.g.,within about 50 cM of these loci). The invention also provideschromosomal QTL intervals that correlate with Charcoal Rot DroughtComplex tolerance. These intervals are located on linkage groups C2, E,B2, G, H, B1, C1, D1b and N. Any marker located within these intervalsalso finds use as a marker for Charcoal Rot Drought Complex toleranceand is also a feature of the invention. These intervals include:

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1);    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); or,    -   (ix) Sat_306 and A363_3 (LG-N).        A plurality of marker loci can be selected in the same plant.        Which QTL markers are selected in combination is not        particularly limited. The QTL markers used in combinations can        be any of the markers listed in FIG. 1, any other marker that is        linked to the markers in FIG. 1 (e.g., the linked markers as        determined from FIG. 6 or determined from the SOYBASE resource),        or any marker within the QTL intervals described herein.

One aspect relates to a method of identifying a first soybean plant orgermplasm that displays tolerance, improved tolerance, or susceptibilityto Charcoal Rot Drought Complex, the method comprising detecting in thefirst soybean plant or germplasm at least one allele of a first markerlocus that is associated with the tolerance, improved tolerance orsusceptibility, wherein the first marker locus localizes within achromosome interval flanked by and including Satt286 and Satt371. In oneaspect, the first marker locus localizes within a chromosomal intervalflanked by and including Satt205 and Satt433. In another aspect, thefirst marker locus localizes within a chromosomal interval flanked byand including Sat_238 and Sat_252. In another aspect, the first markerlocus localizes within a chromosomal interval flanked by and includingSatt307 and A538_1. In yet another aspect, the first marker locuslocalizes within a chromosomal interval flanked by and including Satt307and Satt202. In other aspects, the first marker locus localizes within achromosomal interval flanked by and including, and linked to the Sct_028marker, e.g., Satt286 and Satt202, Satt286 and Sat_252, Satt286 andSatt316, Satt286 and Satt433, Satt286 and Satt371, Sat_402 and Satt202,Sat_402 and Sat_252, Sat_402 and Satt316, Sat_402 and Satt433, Sat_402and Satt371, Satt277 and Satt202, Satt277 and Sat_252, Satt277 andSatt316, Satt277 and Satt433, Satt277 and Satt371, Satt365 and Satt202,Satt365 and Sat_252, Satt365 and Satt316, Satt365 and Satt433, Satt365and Satt371, Satt205 and Satt202, Satt205 and Sat_252, Satt205 andSatt316, Satt205 and Satt433, Satt205 and Satt371, Satt557 and Satt202,Satt557 and Sat_252, Satt557 and Satt316, Satt557 and Satt433, Satt557and Satt371, Satt289 and Satt202, Satt289 and Sat_252, Satt289 andSatt316, Satt289 and Satt433, Satt289 and Satt371, Satt134 and Satt202,Satt134 and Sat_252, Satt134 and Satt316, Satt134 and Satt433, Satt134and Satt371, Sat_312 and Satt202, Sat_312 and Sat_252, Sat_312 andSatt316, Sat_312 and Satt433, Sat_312 and Satt371, Satt489 and Satt202,Satt489 and Sat_252, Satt489 and Satt316, Satt489 and Satt433, Satt489and Satt371, Satt319 and Satt202, Satt319 and Sat_252, Satt319 andSatt316, Satt319 and Satt433, Satt319 and Satt371, Satt658 and Satt202,Satt658 and Sat_252, Satt658 and Satt316, Satt658 and Satt433, Satt658and Satt371, AG36 and Satt202, AG36 and Sat_252, AG36 and Satt316, AG36and Satt433, AG36 and Satt371, Satt100 and Satt202, Satt100 and Sat_252,Satt100 and Satt316, Satt100 and Satt433, Satt100 and Satt371, Sat_251and Satt202, Sat_251 and Sat_252, Sat_251 and Satt316, Sat_251 andSatt433, Sat_251 and Satt371, Sat_142 and Satt202, Sat_142 and Sat_252,Sat_142 and Satt316, Sat_142 and Satt433, Sat_142 and Satt371, Satt708and Satt202, Satt708 and Sat_252, Satt708 and Satt316, Satt708 andSatt433, Satt708 and Satt371, Sat_238 and Satt202, Sat_238 and Sat_252,Sat_238 and Satt316, Sat_238 and Satt433, Sat_238 and Satt371, Satt460and Satt202, Satt460 and Sat_252, Satt460 and Satt316, Satt460 andSatt433, Satt460 and Satt371, Satt079 and Satt202, Satt079 and Sat_252,Satt079 and Satt316, Satt079 and Satt433, Satt079 and Satt371, Sat_263and Satt202, Sat_263 and Sat_252, Sat_263 and Satt316, Sat_263 andSatt433, Sat_263 and Satt371, Staga001 and Satt202, Staga001 andSat_252, Staga001 and Satt316, Staga001 and Satt433, Staga001 andSatt371, Satt307 and Satt202, Satt307 and Sat_252, Satt307 and Satt316,Satt307 and Satt433, and Satt307 and Satt371. In some aspects, the firstmarker locus localizes within a chromosomal interval flanked by andincluding, and closely linked to the Sct_028 marker, e.g., Satt205 andSatt202, Satt205 and Sat_252, Satt205 and Satt316, Satt205 and Satt433,Satt557 and Satt202, Satt557 and Sat_252, Satt557 and Satt316, Satt557and Satt433, Satt289 and Satt202, Satt289 and Sat_252, Satt289 andSatt316, Satt289 and Satt433, Satt134 and Satt202, Satt134 and Sat_252,Satt134 and Satt316, Satt134 and Satt433, Sat_312 and Satt202, Sat_312and Sat_252, Sat_312 and Satt316, Sat_312 and Satt433, Satt489 andSatt202, Satt489 and Sat_252, Satt489 and Satt316, Satt489 and Satt433,Satt319 and Satt202, Satt319 and Sat_252, Satt319 and Satt316, Satt319and Satt433, Satt658 and Satt202, Satt658 and Sat_252, Satt658 andSatt316, Satt658 and Satt433, AG36 and Satt202, AG36 and Sat_252, AG36and Satt316, AG36 and Satt433, Satt100 and Satt202, Satt100 and Sat_252,Satt100 and Satt316, Satt100 and Satt433, Sat_251 and Satt202, Sat_251and Sat_252, Sat_251 and Satt316, Sat_251 and Satt433, Sat_142 andSatt202, Sat_142 and Sat_252, Sat_142 and Satt316, Sat_142 and Satt433,Satt708 and Satt202, Satt708 and Sat_252, Satt708 and Satt316, Satt708and Satt433, Sat_238 and Satt202, Sat_238 and Sat_252, Sat_238 andSatt316, Sat_238 and Satt433, Satt460 and Satt202, Satt460 and Sat_252,Satt460 and Satt316, Satt460 and Satt433, Satt079 and Satt202, Satt079and Sat_252, Satt079 and Satt316, Satt079 and Satt433, Sat_263 andSatt202, Sat_263 and Sat_252, Sat_263 and Satt316, Sat_263 and Satt433,Staga001 and Satt202, Staga001 and Sat_252, Staga001 and Satt316,Staga001 and Satt433, Satt307 and Satt202, Satt307 and Sat_252, Satt307and Satt316, and Satt307 and Satt433. In a preferred embodiment, thefirst marker locus is Sct_028. In another preferred embodiment, the atleast one allele comprises Sct_028: allele-3.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt286 and Satt371 can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of at least one additional marker locus that is associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast one additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt575 and Sat_136, Satt467 and Satt416, Satt612 andA681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt575 and Sat_136,Satt467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 andSat_331, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt575 and Sat_136, Satt467 and Satt416, Satt612 andA681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In further aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least four additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least four additional marker locilocalize within the chromosome intervals flanked by and includingSatt575 and Sat_136, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 andA519_2, and Sat_306 and A363_3. In still further aspects, these methodscan further comprise detecting in the first soybean plant or germplasmat least one allele of each of at least five additional marker loci thatare associated with the tolerance, improved tolerance or susceptibility,wherein the at least five additional marker loci localize within thechromosome intervals flanked by and including Satt575 and Sat_136,Satt467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 andSat_331, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In still further aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least six additional marker loci that are associated with thetolerance, improved tolerance or susceptibility, wherein the at leastsix additional marker loci localize within the chromosome intervalsflanked by and including Satt575 and Sat_136, Satt467 and Satt416,Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3. In still furtheraspects, these methods can further comprise detecting in the firstsoybean plant or germplasm at least one allele of each of at least sevenadditional marker loci that are associated with the tolerance, improvedtolerance or susceptibility, wherein the at least seven additionalmarker loci localize within the chromosome intervals flanked by andincluding Satt575 and Sat_136, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1and A519_2, and Sat_306 and A363_3. In still further aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least eight additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least eight additional marker locilocalize within the chromosome intervals flanked by and includingSatt575 and Sat_136, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 andA519_2, and Sat_306 and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt286 and Satt371, in some aspects,the at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects. In one aspect, the introgressed soybean plant orgermplasm comprises Sct_028:allele-3.

Another aspect relates to a method of identifying a first soybean plantor germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Satt575 andSat_136. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Satt411 and Satt720. Inanother aspect, the first marker locus localizes within a chromosomalinterval flanked by and including Sat_124 and A963_1. In yet anotheraspect, the first marker locus localizes within a chromosomal intervalflanked by and including Sat_124 and Satt384. In other aspects, thefirst marker locus localizes within a chromosomal interval flanked byand including, and linked to the Satt512 marker, e.g., Satt575 andSatt384, Satt575 and Satt691, Satt575 and Satt720, Satt575 and Satt651,Satt575 and Satt212, Satt575 and Satt598, Satt575 and Satt573, Satt575and Sat_136, Satt213 and Satt384, Satt213 and Satt691, Satt213 andSatt720, Satt213 and Satt651, Satt213 and Satt212, Satt213 and Satt598,Satt213 and Satt573, Satt213 and Sat_136, Sat_112 and Satt384, Sat_112and Satt691, Sat_112 and Satt720, Sat_112 and Satt651, Sat_112 andSatt212, Sat_112 and Satt598, Sat_112 and Satt573, Sat_112 and Sat_136,Satt411 and Satt384, Satt411 and Satt691, Satt411 and Satt720, Satt411and Satt651, Satt411 and Satt212, Satt411 and Satt598, Satt411 andSatt573, Satt411 and Sat_136, Sat_124 and Satt384, Sat_124 and Satt691,Sat¹²⁴ and Satt720, Sat_124 and Satt651, Sat_124 and Satt212, Sat_124and Satt598, Sat_124 and Satt573, Sat_124 and Sat_136, In some aspects,the first marker locus localizes within a chromosomal interval flankedby and including, and closely linked to the Satt512 marker, e.g.,Satt411 and Satt384, Satt411 and Satt691, Satt411 and Satt720, Sat_124and Satt384, Sat_124 and Satt691, and Sat_124 and Satt720. In apreferred embodiment, the first marker locus is Satt512. In anotherpreferred embodiment, the at least one allele comprises Satt512:allele-2or Satt512:allele-5.

Methods wherein the first marker localizes within a chromosomal intervalflanked by and including Satt575 and Sat_136 can further comprisedetecting in the first soybean plant or germplasm at least one allele ofat least one additional marker locus that is associated with thetolerance, improved tolerance or susceptibility, wherein the at leastone additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Sat467 and Satt416, Satt612 andA681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Sat467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 andSat_331, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Sat467 and Satt416, Satt612 andA681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least four additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Sat467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_306 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least five additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least five additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Sat467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastsix additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least sixadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Sat467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 andA519_2, and Sat_306 and A363_3. In additional aspects, these methods canfurther comprise detecting in the first soybean plant or germplasm atleast one allele of each of at least seven additional marker loci thatare associated with the tolerance, improved tolerance or susceptibility,wherein the at least seven additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Sat467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leasteight additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least eightadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Sat467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 andA519_2, and Sat_306 and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt575 and Sat_136, in some aspects,the at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects. In one aspect, the introgressed soybean plant orgermplasm comprises Satt512:allele-2 or Satt512:allele-5.

A further aspect relates to a method of identifying a first soybeanplant or germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Satt467 andSatt416. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Satt126 and Sct_034. Inanother aspect, the first marker locus localizes with a chromosomalinterval flanked by and including RGA_8 and A343_1. In yet anotheraspect, the first marker locus localizes within a chromosomal intervalflanked by and including Sat_287 and OP_T02. In still another aspect,the first marker locus localizes within a chromosomal interval flankedby and including Sat_287 and Sct_034. In other aspects, the first markerlocus localizes within a chromosomal interval flanked by and including,and linked to the S60211-TB marker, e.g., Satt467 and Sct_034, Satt467and Satt083, Satt467 and Satt168, Satt467 and Satt416, Sat_342 andSct_034, Sat_342 and Satt083, Sat_342 and Satt168, Sat_342 and Satt416,Satt126 and Sct_034, Satt126 and Satt083, Satt126 and Satt168, Satt126and Satt416, Sat_287 and Sct_034, Sat_287 and Satt083, Sat_287 andSatt168, and Sat_287 and Satt416. In some aspects, the first markerlocus localizes within a chromosomal interval flanked by and including,and closely linked to the S60211-TB marker, e.g., Satt126 and A343_1,Satt126 and OP_T02, Satt126 and B142_1, Sat_287 and A343_1, Sat_287 andOP_T02, and Sat_287 and B142_1. In a preferred embodiment, the firstmarker locus is S60211-TB. In another preferred embodiment, the at leastone allele comprises S60211-TB:allele-1.

Methods wherein the first marker locus localizes within a chromosomeinterval flanked by and including Satt467 and Satt416 can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of at least one additional marker locus that is associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast one additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Satt575 and Sat_136, Satt612 andA681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Sat_136, Satt612 and A681_1, Sat_158 and A162_1, Satt444 andSat_331, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Sat_136, Satt612 andA681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least four additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Sat_136, Satt612 and A681_1, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_306 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least five additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least five additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSat_136, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastsix additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least sixadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Sat_136, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 andA519_2, and Sat_306 and A363_3. In additional aspects, these methods canfurther comprise detecting in the first soybean plant or germplasm atleast one allele of each of at least seven additional marker loci thatare associated with the tolerance, improved tolerance or susceptibility,wherein the at least seven additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSat_136, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leasteight additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least eightadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Sat_136, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 andA519_2, and Sat_306 and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt467 and Satt416, in some aspects,the at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects. In one aspect, the introgressed soybean plant orgermplasm comprises S60211-TB:allele-1.

Another aspect relates to a method of identifying a first soybean plantor germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Satt612 andA681_1. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Satt612 and Sat_064. Inone aspect, the first marker locus localizes within a chromosomalinterval flanked by and including Sct_199 and Sat_064. In anotheraspect, the first marker locus localizes with a chromosomal intervalflanked by and including L154_1 and A690_2. In yet another aspect, thefirst marker localizes within a chromosomal interval flanked by andincluding Satt191 and Sct_187. In still another aspect, the first markerlocalizes within a chromosomal interval flanked by and including Satt472and Sct_187. In other aspects, the first marker locus localizes within achromosomal interval flanked by and including, and linked to the Sat_117marker or the S01954-1-A marker, e.g., Satt612 and A690_2, Satt612 andBng069_1, Satt612 and Sct_187, Satt612 and Sat_372, Satt612 and Sat_064,Satt612 and A681_1, AF162283 and A690_2, AF162283 and Bng069_1, AF162283and Sct_187, AF162283 and Sat_372, AF162283 and Sat_064, AF162283 andA681_1, Sct_199 and A690_1, Sct_199 and Bng069_1, Sct_199 and Sct_187,Sct_199 and Sat_372, Sct_199 and Sat_064, Sct_199 and A681_1, Satt472and A690_1, Satt472 and Bng069_1, Satt472 and Sct_187, Satt472 andSat_372, Satt472 and Sat_064, Satt472 and A681_1, Satt191 and A690_1,Satt191 and Bng069_1, Satt191 and Sct_187, Satt191 and Sat_372, Satt191and Sat_064, and Satt191 and A681_1. In some aspects, the first markerlocus localizes with a chromosomal interval flanked by and including,and closely linked to the Sat_117 marker or the S01954-1-A marker, e.g.,Sct_199 and A690_1, Sct_199 and Bng069_1, Sct_199 and Sct_187, Sct_199and Sat_372, Sct_199 and Sat_064, Satt472 and A690_1, Satt472 andBng069_1, Satt472 and Sct_187, Satt472 and Sat_372, Satt472 and Sat_064,Satt191 and A690_1, Satt191 and Bng069_1, Satt191 and Sct_187, Satt191and Sat_372, and Satt191 and Sat_064. In a preferred embodiment, thefirst marker locus is Sat_117. In another preferred embodiment, the atleast one allele comprises Sat_117: allele-2.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt288 and A681_1, or Satt612 andA681_1, can further comprise detecting in the first soybean plant orgermplasm at least one allele of at least one additional marker locusthat is associated with the tolerance, improved tolerance orsusceptibility, wherein the at least one additional marker locuslocalizes within a chromosome interval flanked by and including achromosomal interval selected from the group consisting of Satt286 andSatt371, Satt575 and Sat_136, Satt467 and Satt416, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_307 and A363_3. In some aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least two additional marker loci that are associated with thetolerance, improved tolerance or susceptibility, wherein the at leasttwo additional marker loci localize within chromosome intervals flankedby and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Sat_136, Satt467 andSatt416, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_307 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least three additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least three additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Sat_136, Satt467 and Satt416, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_307 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least four additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least four additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSat_136, Satt467 and Satt416, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_307 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastfive additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least fiveadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Sat_136, Satt467 and Satt416,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1and A519_2, and Sat_307 and A363_3. In additional aspects, these methodscan further comprise detecting in the first soybean plant or germplasmat least one allele of each of at least six additional marker loci thatare associated with the tolerance, improved tolerance or susceptibility,wherein the at least six additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSat_136, Satt467 and Satt416, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_307 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastseven additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least sevenadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Sat_136, Satt467 and Satt416,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1and A519_2, and Sat_307 and A363_3. In additional aspects, these methodscan further comprise detecting in the first soybean plant or germplasmat least one allele of each of at least eight additional marker locithat are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least eight additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Sat_136, Satt467 and Satt416, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_307 and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt612 and A681_1, in some aspects,the at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects. In one aspect, the introgressed soybean plant orgermplasm comprises Sat_117: allele-2.

A further aspect relates to a method of identifying a first soybeanplant or germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Sat_158 andA162_1. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Satt637 and Satt434. Inanother aspect, the first marker locus localizes with a chromosomalinterval flanked by and including Satt181 and Sat_218. In yet anotheraspect, the first marker locus localizes with a chromosomal intervalflanked by and including Satt181 and A810_1. In other aspects, the firstmarker locus localizes within a chromosomal interval flanked by andincluding, and linked to the P13158A marker, e.g., Sat_158 and Sat_218,Sat_158 and Sat_180, Sat_158 and Satt434, Sat_158 and A162_1, Satt302and Sat_218, Satt302 and Sat_180, Satt302 and Satt434, Satt302 andA162_1, Sat_175 and Sat_218, Sat_175 and Sat_180, Sat_175 and Satt434,Sat_175 and A162_1, Sat_216 and Sat_218, Sat_216 and Sat_180, Sat_216and Satt434, Sat_216 and A162_1, Satt637 and Sat_218, Satt637 andSat_180, Satt637 and Satt434, Satt637 and A162_1, Satt142 and Sat_218,Satt142 and Sat_180, Satt142 and Satt434, Satt142 and A162_1, Satt293and Sat_218, Satt293 and Sat_180, Satt293 and Satt434, Satt293 andA162_1, Satt317 and Sat_218, Satt317 and Sat_180, Satt317 and Satt434,Satt317 and A162_1, Satt181 and Sat_218, Satt181 and Sat_180, Satt181and Satt434, Satt181 and A162_1. In some aspects, the first marker locuslocalizes within a chromosomal interval flanked by and including, andclosely linked to the P13158A marker, e.g., Sat_181 and A810_1, andSat_181 and Sat_218. In a preferred embodiment, the first marker locusis P13158A. In another preferred embodiment, the at least one allelecomprises P13158A:allele-2.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Sat_158 and A162_1 can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of at least one additional marker locus that is associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast one additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Satt444and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Satt444 and Sat_331, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least four additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_306 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least five additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least five additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSatt1362, Satt467 and Satt416, Satt612 and A681_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastsix additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least sixadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Satt1362, Satt467 and Satt416,Satt612 and A681_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1and A519_2, and Sat_306 and A363_3. In additional aspects, these methodscan further comprise detecting in the first soybean plant or germplasmat least one allele of each of at least seven additional marker locithat are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least seven additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1 and A519_2, andSat_306 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least eight additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least eight additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSatt1362, Satt467 and Satt416, Satt612 and A681_1, Satt444 and Sat_331,Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Sat_158 and A162_1, in some aspectsthe at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects. In one aspect, the introgressed soybean plant orgermplasm comprises P13158A:allele-2.

A further aspect relates to a method of identifying a first soybeanplant or germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Satt444 andSat_331. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Sat_123 and Satt484. Inanother aspect, the first marker locus localizes with a chromosomalinterval flanked by and including Satt359 and Satt484. In yet anotheraspect, the first marker locus localizes with a chromosomal intervalflanked by and including Satt359 and R244_1. In other aspects, the firstmarker locus localizes within a chromosomal interval flanked by andincluding, and linked to the 563880-CB marker, e.g., Satt444 andSatt484, Satt444 and Satt453, Satt444 and Sat_331, Satt665 and Satt484,Satt665 and Satt453, Satt665 and Sat_331, Sat_123 and Satt484, Sat_123and Satt453, Sat_123 and Sat_331, Satt359 and Satt484, Satt359 andSatt453, and Satt359 and Sat_331. In some aspects, the first markerlocus localizes within a chromosomal interval flanked by and including,and closely linked to the 563880-CB marker, e.g., Sat_123 and Satt484,and Satt359 and Satt484. In a preferred embodiment, the first markerlocus is 563880-CB.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt444 and Sat_331 can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of at least one additional marker locus that is associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast one additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least four additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306and A363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least five additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast five additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least six additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least six additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least seven additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast seven additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Bng019_1 and Sct_191,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least eight additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least eight additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Bng019_1 and Sct_191, A605_1 and A519_2, and Sat_306and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Satt444 and Sat_331, in some aspectsthe at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects.

A further aspect relates to a method of identifying a first soybeanplant or germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Bng019_1 andSct_191. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Satt578 and Satt294. Inanother aspect, the first marker locus localizes with a chromosomalinterval flanked by and including Satt607 and G214_24. In yet anotheraspect, the first marker locus localizes with a chromosomal intervalflanked by and including Dia and L192_1. In other aspects, the firstmarker locus localizes within a chromosomal interval flanked by andincluding, and linked to the S00415-1-A marker, e.g., Satt578 andSat_311, Satt578 and Satt670, Satt578 and Sat_476, Satt578 and Sat399,Satt578 and Satt139, Satt578 and Satt718, Satt607 and Sat_311, Satt607and Satt670, Satt607 and Sat_476, Satt607 and Sat399, Satt607 andSatt139, Satt607 and Satt718, Satt646 and Sat_311, Satt646 and Satt670,Satt646 and Sat_476, Satt646 and Sat399, Satt646 and Satt139, Satt646and Satt718, Dia and Sat_311, Dia and Satt670, Dia and Sat_476, Dia andSat399, Dia and Satt139, and Dia and Satt718. In some aspects, the firstmarker locus localizes within a chromosomal interval flanked by andincluding, and closely linked to the S00415-1-A marker, e.g., Satt607and Sat399, Satt607 and Satt139, Satt607 and Satt718, Satt646 andSat399, Satt646 and Satt139, Satt646 and Satt718, Dia and Sat399, Diaand Satt139, and Dia and Satt718. In a preferred embodiment, the firstmarker locus is S00415-1-A.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Bng019_1 and Sct_191 can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of at least one additional marker locus that is associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast one additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,A605_1 and A519_2, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least four additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, A605_1 and A519_2, and Sat_306and A363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least five additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast five additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,A605_1 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least six additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least six additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A1621, Satt444 and Sat_331, A605_1 and A519_2, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least seven additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast seven additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A1621, Satt444 and Sat_331,A6051 and A519_2, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least eight additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least eight additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A1621, Satt444 and Sat_331, A605_1 and A519_2, and Sat_306and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Bng019_1 and Sct_191, in some aspectsthe at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects.

A further aspect relates to a method of identifying a first soybeanplant or germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including A605_1 and A519_2.In one aspect, the first marker locus localizes within a chromosomalinterval flanked by and including Satt360 and Sat_139. In anotheraspect, the first marker locus localizes with a chromosomal intervalflanked by and including Satt428 and B194_2. In yet another aspect, thefirst marker locus localizes with a chromosomal interval flanked by andincluding Satt644 and Satt041. In other aspects, the first marker locuslocalizes within a chromosomal interval flanked by and including, andlinked to the S00705-1-A marker, e.g., Sat_423 and Sat_069, Sat_423 andSat_139, Sat_423 and B194_2, Sat_423 and Satt041, Satt290 and Sat_069,Satt290 and Sat_139, Satt290 and B194_2, Satt290 and Satt041, Satt005and Sat_069, Satt005 and Sat_139, Satt005 and B194_2, Satt005 andSatt041, Satt579 and Sat_069, Satt579 and Sat_139, Satt579 and B194_2,Satt579 and Satt041, ∥Satt428 and Sat_069, Satt428 and Sat_139, Satt428and B194_2, Satt428 and Satt041, Satt644 and Sat_069, Satt644 andSat_139, Satt644 and B194_2, and Satt644 and Satt041. In some aspects,the first marker locus localizes within a chromosomal interval flankedby and including, and closely linked to the S00705-1-A marker, e.g.,Satt428 and B194_2, Satt428 and Satt041, Satt644 and B194_2, and Satt644and Satt041. In a preferred embodiment, the first marker locus isS00705-1-A.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including A605_1 and A519_2 can further comprisedetecting in the first soybean plant or germplasm at least one allele ofat least one additional marker locus that is associated with thetolerance, improved tolerance or susceptibility, wherein the at leastone additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, and Sat_306 and A363_3. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, and Sat_306 andA363_3. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, and Sat_306 and A363_3. In additional aspects,these methods can further comprise detecting in the first soybean plantor germplasm at least one allele of each of at least four additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, andSat_306 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least five additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least five additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSatt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, and Sat_306 and A363_3. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastsix additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least sixadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Satt1362, Satt467 and Satt416,Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1and Sct_191, and Sat_306 and A363_3. In additional aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least seven additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least seven additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, andSat_306 and A363_3. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least eight additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least eight additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSatt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, and Sat_306 and A363_3.

In methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including A605_1 and A519_2, in some aspects theat least one allele is correlated with tolerance or improved tolerance,and the method further comprises introgressing the allele in the firstsoybean plant or germplasm into a second soybean plant or germplasm toproduce an introgressed soybean plant or germplasm. Introgressed soybeanplants or germplasm produced by these methods are additional aspects.

A further aspect relates to a method of identifying a first soybeanplant or germplasm that displays tolerance, improved tolerance, orsusceptibility to Charcoal Rot Drought Complex, the method comprisingdetecting in the first soybean plant or germplasm at least one allele ofa first marker locus that is associated with the tolerance, improvedtolerance or susceptibility, wherein the first marker locus localizeswithin a chromosome interval flanked by and including Sat_306 andA363_3. In one aspect, the first marker locus localizes within achromosomal interval flanked by and including Sat_295 and A363_3. Inanother aspect, the first marker locus localizes with a chromosomalinterval flanked by and including Satt022 and A363_3. In yet anotheraspect, the first marker locus localizes with a chromosomal intervalflanked by and including Sat_125 and A455_2. In other aspects, the firstmarker locus localizes within a chromosomal interval flanked by andincluding, and linked to the S02118-1-A marker, e.g., Sat_306 andA455_2, Sat_295 and A455_2, Satt022 and A455_2, and Sat_125 and A363_3.In some aspects, the first marker locus localizes within a chromosomalinterval flanked by and including, and closely linked to the S02118-1-Amarker, e.g., Satt022 and A455_2, Satt022 and A363_3, Sat_125 andA455_2, and Sat_125 and A363_3. In a preferred embodiment, the firstmarker locus is S02118-1-A.

Methods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Sat_306 and A363_3 can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of at least one additional marker locus that is associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast one additional marker locus localizes within a chromosome intervalflanked by and including a chromosomal interval selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, and A605_1 and A519_2. In some aspects, thesemethods can further comprise detecting in the first soybean plant orgermplasm at least one allele of each of at least two additional markerloci that are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least two additional marker loci localizewithin chromosome intervals flanked by and including chromosomalintervals selected from the group consisting of Satt286 and Satt371,Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, and A605_1 andA519_2. In additional aspects, these methods can further comprisedetecting in the first soybean plant or germplasm at least one allele ofeach of at least three additional marker loci that are associated withthe tolerance, improved tolerance or susceptibility, wherein the atleast three additional marker loci localize within chromosome intervalsflanked by and including chromosomal intervals selected from the groupconsisting of Satt286 and Satt371, Satt575 and Satt1362, Satt467 andSatt416, Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331,Bng019_1 and Sct_191, and A605_1 and A519_2. In additional aspects,these methods can further comprise detecting in the first soybean plantor germplasm at least one allele of each of at least four additionalmarker loci that are associated with the tolerance, improved toleranceor susceptibility, wherein the at least four additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, andA605_1 and A519_2. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least five additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least five additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSatt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, and A605_1 and A519_2. Inadditional aspects, these methods can further comprise detecting in thefirst soybean plant or germplasm at least one allele of each of at leastsix additional marker loci that are associated with the tolerance,improved tolerance or susceptibility, wherein the at least sixadditional marker loci localize within chromosome intervals flanked byand including chromosomal intervals selected from the group consistingof Satt286 and Satt371, Satt575 and Satt1362, Satt467 and Satt416,Satt612 and A681_1, Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1and Sct_191, and A605_1 and A519_2. In additional aspects, these methodscan further comprise detecting in the first soybean plant or germplasmat least one allele of each of at least seven additional marker locithat are associated with the tolerance, improved tolerance orsusceptibility, wherein the at least seven additional marker locilocalize within chromosome intervals flanked by and includingchromosomal intervals selected from the group consisting of Satt286 andSatt371, Satt575 and Satt1362, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, andA605_1 and A519_2. In additional aspects, these methods can furthercomprise detecting in the first soybean plant or germplasm at least oneallele of each of at least eight additional marker loci that areassociated with the tolerance, improved tolerance or susceptibility,wherein the at least eight additional marker loci localize withinchromosome intervals flanked by and including chromosomal intervalsselected from the group consisting of Satt286 and Satt371, Satt575 andSatt1362, Satt467 and Satt416, Satt612 and A681_1, Sat_158 and A162_1,Satt444 and Sat_331, Bng019_1 and Sct_191, and A605_1 and A519_2. Inmethods wherein the first marker locus localizes within a chromosomalinterval flanked by and including Sat_306 and A363_3, in some aspectsthe at least one allele is correlated with tolerance or improvedtolerance, and the method further comprises introgressing the allele inthe first soybean plant or germplasm into a second soybean plant orgermplasm to produce an introgressed soybean plant or germplasm.Introgressed soybean plants or germplasm produced by these methods areadditional aspects.

An additional aspect relates to a method of selecting at least onesoybean plant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Satt286 andSatt371 on linkage group C2, said method comprising (a) testing at leastone marker on said chromosomal interval for said quantitative traitlocus; and (b) selecting said soybean plant comprising said quantitativetrait locus. Other chromosomal intervals, such as those described aboveor elsewhere herein, are also useful in these methods.

Another aspect relates to a method of selecting at least one soybeanplant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Satt575 andSat_136 on linkage group E, said method comprising (a) testing at leastone marker on said chromosomal interval for said quantitative traitlocus; and (b) selecting said soybean plant comprising said quantitativetrait locus. Other chromosomal intervals, such as those described aboveor elsewhere herein, are also useful in these methods.

A further aspect relates to a method of selecting at least one soybeanplant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Satt467 andSatt416 on linkage group B2, said method comprising (a) testing at leastone marker on said chromosomal interval for said quantitative traitlocus; and (b) selecting said soybean plant comprising said quantitativetrait locus. Other chromosomal intervals, such as those described aboveor elsewhere herein, are also useful in these methods.

Another aspect relates to a method of selecting at least one soybeanplant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Satt612 and A681_1on linkage group G, said method comprising (a) testing at least onemarker on said chromosomal interval for said quantitative trait locus;and (b) selecting said soybean plant comprising said quantitative traitlocus. Other chromosomal intervals, such as those described above orelsewhere herein, are also useful in these methods.

Yet another aspect relates to a method of selecting at least one soybeanplant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Satt612 andSat_064 on linkage group G, said method comprising (a) testing at leastone marker on said chromosomal interval for said quantitative traitlocus; and (b) selecting said soybean plant comprising said quantitativetrait locus. Other chromosomal intervals, such as those described aboveor elsewhere herein, are also useful in these methods.

An additional aspect relates to a method of selecting at least onesoybean plant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Sat_158 and A162_1on linkage group H, said method comprising (a) testing at least onemarker on said chromosomal interval for said quantitative trait locus;and (b) selecting said soybean plant comprising said quantitative traitlocus. Other chromosomal intervals, such as those described above orelsewhere herein, are also useful in these methods.

An additional aspect relates to a method of selecting at least onesoybean plant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Sat_158 and A162_1on linkage group H, said method comprising (a) testing at least onemarker on said chromosomal interval for said quantitative trait locus;and (b) selecting said soybean plant comprising said quantitative traitlocus. Other chromosomal intervals, such as those described above orelsewhere herein, are also useful in these methods.

Yet another aspect relates to a method of selecting at least one soybeanplant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Satt444 andSat_331 on linkage group B1, said method comprising (a) testing at leastone marker on said chromosomal interval for said quantitative traitlocus; and (b) selecting said soybean plant comprising said quantitativetrait locus. Other chromosomal intervals, such as those described aboveor elsewhere herein, are also useful in these methods.

A further additional aspect relates to a method of selecting at leastone soybean plant by marker assisted selection of a quantitative traitlocus associated with tolerance or improved tolerance to Charcoal RotDrought Complex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Bng019_1 andSct_191 on linkage group C1, said method comprising (a) testing at leastone marker on said chromosomal interval for said quantitative traitlocus; and (b) selecting said soybean plant comprising said quantitativetrait locus. Other chromosomal intervals, such as those described aboveor elsewhere herein, are also useful in these methods.

A further additional aspect relates to a method of selecting at leastone soybean plant by marker assisted selection of a quantitative traitlocus associated with tolerance or improved tolerance to Charcoal RotDrought Complex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers A605_1 and A519_2on linkage group D1 b, said method comprising (a) testing at least onemarker on said chromosomal interval for said quantitative trait locus;and (b) selecting said soybean plant comprising said quantitative traitlocus. Other chromosomal intervals, such as those described above orelsewhere herein, are also useful in these methods.

A further additional aspect relates to a method of selecting at leastone soybean plant by marker assisted selection of a quantitative traitlocus associated with tolerance or improved tolerance to Charcoal RotDrought Complex, wherein said quantitative trait locus is localized to achromosomal interval defined by and including markers Sat_306 and A363_3on linkage group N, said method comprising (a) testing at least onemarker on said chromosomal interval for said quantitative trait locus;and (b) selecting said soybean plant comprising said quantitative traitlocus. Other chromosomal intervals, such as those described above orelsewhere herein, are also useful in these methods.

Further aspects relate to methods of selecting at least one soybeanplant by marker assisted selection of a quantitative trait locusassociated with tolerance or improved tolerance to Charcoal Rot DroughtComplex, wherein said quantitative trait locus is localized to at leastone of (i) a chromosomal interval defined by and including markersSatt286 and Satt371 on linkage group C2, (ii) a chromosomal intervaldefined by and including markers Satt575 and Sat_136 on linkage group E,(iii) a chromosomal interval defined by and including markers Satt467and Satt416 on linkage group B2, (iv) a chromosomal interval defined byand including markers Satt612 and A681_1 on linkage group G, (v) achromosomal interval defined by and including markers Satt612 andSat_064 on linkage group G, (vi) a chromosomal interval defined by andincluding markers Sat_158 and A162_1 on linkage group H, (vii) achromosomal interval defined by and including markers Sat_158 andSatt434 on linkage group H, (viii) a chromosomal interval defined by andincluding markers Satt444 and Sat_331 on linkage group B1, (ix) achromosomal interval defined by and including markers Bng019_1 andSct_191 on linkage group C1, (x) a chromosomal interval defined by andincluding markers Satt578 and Sct_191 on linkage group C1, (xi) achromosomal interval defined by and including markers A605_1 and A519_2on linkage group D1 b, (xii) a chromosomal interval defined by andincluding markers Sat_423 and Sat_069 on linkage group D1 b, and (xiii)a chromosomal interval defined by and including markers Sat_306 andA363_3 on linkage group N, said method comprising: (a) testing at leastone maker on each of said chromosomal intervals for said quantitativetrait locus; and (b) selecting said soybean plant comprising saidquantitative trait locus. Other chromosomal intervals, such as thosedescribed above or elsewhere herein, are also useful in these methods.

Additional aspects relate to methods of producing a soybean plant havingtolerance or improved tolerance to Charcoal Rot Drought Complex, themethod comprising introducing an exogenous nucleic acid into a targetsoybean plant or progeny thereof, wherein the exogenous nucleic acid isderived from a nucleotide sequence that is linked to at least onefavorable allele of a marker locus that is associated with tolerance orimproved tolerance to Charcoal Rot Drought Complex, wherein the markerlocus localizes within a chromosomal interval selected from the groupconsisting of chromosomal intervals flanked by and including Satt286 andSatt371, Satt575 and Sat_136, Satt467 and Satt416, Satt612 and A681_1,Sat_158 and A162_1, Satt444 and Sat_331, Bng019_1 and Sct_191, A605_1and A519_2, and Sat_306 and A363_3; whereby the resulting transgenicplant displays tolerance or improved tolerance to Charcoal Rot DroughtComplex. Other chromosomal intervals, such as those described above orelsewhere herein, are also useful in these methods.

The markers that are linked to the QTL markers of FIG. 1 can be closelylinked, for example, within about 10 cM from the FIG. 1 QTL markers. Insome embodiments, the linked locus displays a genetic recombinationdistance of 9 cM, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25, or lessfrom the QTL marker. In some embodiments, the closely linked locus isselected from the list of marker loci provided in FIG. 6.

In some embodiments, preferred QTL markers are selected from Satt512 onlinkage group E, SCT_028 on linkage group C2, S60211-TB on linkage groupB2, Sat_117 on linkage group G, S01954-1-A on linkage group G, andS00415-1-A on linkage group C1. In other embodiments, preferredfavorable alleles are selected from Satt512:allele-2, Satt512:allele-5,SCT_028: allele-3, S60211-TB:allele-1, and Sat_117: allele-2. Inadditional embodiments, preferred unfavorable alleles are/selected fromSatt512:allele-1, SCT_028: allele-1, and 560211-TB:allele-2.

In some embodiments, the germplasm is a soybean line or variety. In someaspects, the tolerance or improved tolerance is a non-race specifictolerance or a non-race specific improved tolerance. In some aspects,the tolerance, improved tolerance, or susceptibility of a soybean plantto Charcoal Rot Drought Complex can be quantitated using any suitablemeans, for example soybean cultivars may be grown at a field locationwith a known history of Charcoal Rot and rated based on disease symptomsat the appropriate time.

Experienced plant breeders can recognize tolerant soybean plants in thefield, and can select the tolerant individuals or populations forbreeding purposes or for propagation. In this context, the plant breederrecognizes “tolerant” and “non-tolerant,” or “susceptible” soybeanplants.

Such plant breeding practitioners will appreciate that plant toleranceis a phenotypic spectrum consisting of extremes in tolerance,susceptibility and a continuum of intermediate tolerance phenotypes.Tolerance also varies due to environmental effects and the severity ofpathogen infection. Evaluation of phenotypes using reproducible assaysand tolerance scoring methods are of value to scientists who seek toidentify genetic loci that impart tolerance, conduct marker assistedselection for tolerant population, and for introgression techniques tobreed a tolerance trait into an elite soybean line, for example.

Various methods are known in the art for determining (and measuring) thetolerance of a soybean plant to Charcoal Rot Drought Complex. Theydescribe a tolerance measurement scale of 1-9, with 9=no disease and1=total necrosis caused by Macrophomina phaseolina. It will beappreciated that all such scales are relative and that numbering andprecise correlation to any scale can be performed at the discretion ofthe practitioner.

Typically, individual field tests are monitored for Charcoal rotsymptoms during the middle to late vegetative stages, but such symptomstypically appear in the early reproductive stage (during flowering andearly pod set). Data collection is usually done in 3 or 4 successivescorings about 7 days apart. Scorings continue until worsening symptomscan no longer be quantified or until the symptoms are confounded byother factors such as other diseases, insect pressure, severe weather,or advancing maturity.

In general, while there is a certain amount of subjectivity to assigningseverity measurements for disease caused symptoms, assignment to a givenscale as noted above is well within the skill of a practitioner in thefield. Measurements can also be averaged across multiple scorers toreduce variation in field measurements. Furthermore, although protocolsusing artificial inoculation of field nurseries with Macrophominaphaseolina can certainly be used in assessing tolerance, it is alsotypical for tolerance ratings to be based on actual field observationsof fortuitous natural disease incidence, with the informationcorresponding to disease incidence for a cultivar being averaged overmany locations and, typically, several years of crop growing.

If there is no disease present, the rating system above is inapplicable,because everything in an uninfected field scores as tolerant. However,if Charcoal Rot does occur in a specific field location, all of thelines at that location can be scored as noted above. These scores canaccumulate over locations and years to show disease tolerance for givencultivars. Thus, older lines can have more years of observation thannewer ones etc. However, relative measurements can easily be made usingthe scoring system noted above. Furthermore, the tolerance ratings canbe updated and refined each year based on the previous year'sobservations in the field. Based on this, Charcoal Rot scores for acultivar are relative measurements of tolerance.

The experiments described herein score soybean tolerance to Charcoal RotDrought Complex using the following scale: 9=no disease symptoms withnormal plant growth; 8=very slight symptoms including up to a 10%reduction in leaflet and overall canopy size with no wilting; 7=wiltingbeginning to appear at the uppermost two nodes; 6=wilting at theuppermost three nodes and leaflet yellowing beginning appear; 5=Up to 5%plant death with wilting and yellowing of leaflets occurring at theuppermost four nodes; 4=Up to 10% plant death with wilting and yellowingof leaflets occurring at the uppermost four nodes; 3=Up to 25% plantdeath with wilting and yellowing of leaflets occurring at the uppermostfour nodes; 2=up to 50% plant death; 1=50-100% plant death. FIG. 8 givesa representative example of cultivars with vastly different Charcoal RotDrought Complex tolerance using this scoring system.

Any of a variety of techniques can be used to identify a marker allele.It is not intended that the method of allele detection be limited in anyway. Methods for allele detection typically include molecularidentification methods such as amplification and detection of the markeramplicon. For example, an allelic form of a polymorphic simple sequencerepeat (SSR), or of a single nucleotide polymorphism (SNP) can bedetected, e.g., by an amplification based technology. In these and otheramplification based detection methods, the marker locus or a portion ofthe marker locus is amplified (e.g., via PCR, LCR or transcription usinga nucleic acid isolated from a soybean plant of interest as a template)and the resulting amplified marker amplicon is detected. In one exampleof such an approach, an amplification primer or amplification primerpair is admixed with genomic nucleic acid isolated from the firstsoybean plant or germplasm, wherein the primer or primer pair iscomplementary or partially complementary to at least a portion of themarker locus, and is capable of initiating DNA polymerization by a DNApolymerase using the soybean genomic nucleic acid as a template. Theprimer or primer pair (e.g., a primer pair provided in FIG. 2) isextended in a DNA polymerization reaction having a DNA polymerase and atemplate genomic nucleic acid to generate at least one amplicon. In anycase, data representing the detected allele(s) can be transmitted (e.g.,electronically or via infrared, wireless or optical transmission) to acomputer or computer readable medium for analysis or storage. In someembodiments, plant RNA is the template for the amplification reaction.In other embodiments, plant genomic DNA is the template for theamplification reaction. In some embodiments, the QTL marker is a SNPtype marker, and the detected allele is a SNP allele (see, e.g., FIG.3), and the method of detection is allele specific hybridization (ASH).

In some embodiments, the allele that is detected is a favorable allelethat positively correlates with tolerance or improved tolerance.Alternatively, the allele that is detected can be an allele thatcorrelates with disease susceptibility or reduced disease tolerance, andthat allele is counter-selected. For example, alleles that can beselected for (favorable alleles) or against (unfavorable alleles)include:

Favorable Alleles:

Sct_028:allele-3, Satt512:allele-2, Satt512:allele-5,S60211-TB:allele-1, Sat_117: allele-2, P13158A:allele-2.

Unfavorable Alleles:

Sct_028: allele-1, Satt512:allele-1, S60211-TB:allele-2,S63880-CB:allele-3.

In the case where more than one marker is selected, an allele isselected for each of the markers; thus, two or more alleles areselected. In some embodiments, it can be the case that a marker locuswill have more than one advantageous allele, and in that case, eitherallele can be selected.

It will be appreciated that the ability to identify QTL marker loci thatcorrelate with tolerance, improved tolerance, or susceptibility of asoybean plant to Charcoal Rot Drought Complex provides a method forselecting plants that have favorable marker loci as well. That is, anyplant that is identified as comprising a desired marker locus (e.g., amarker allele that positively correlates with tolerance) can be selectedfor, while plants that lack the locus, or that have a locus thatnegatively correlates with tolerance, can be selected against. Thus, inone method, subsequent to identification of a marker locus, the methodsinclude selecting (e.g., isolating) the first soybean plant orgermplasm, or selecting a progeny of the first plant or germplasm. Insome embodiments, the resulting selected first soybean plant orgermplasm can be crossed with a second soybean plant or germplasm (e.g.,an elite or exotic soybean, depending on characteristics that aredesired in the progeny).

Similarly, in other embodiments, if an allele is correlated withtolerance or improved tolerance to Charcoal Rot Drought Complex, themethod can include introgressing the allele into a second soybean plantor germplasm to produce an introgressed soybean plant or germplasm. Insome embodiments, the second soybean plant or germplasm will typicallydisplay reduced tolerance to Charcoal Rot Drought Complex as compared tothe first soybean plant or germplasm, while the introgressed soybeanplant or germplasm will display an increased tolerance to Charcoal RotDrought Complex as compared to the second plant or germplasm. Anintrogressed soybean plant or germplasm produced by these methods isalso a feature of the invention. In some embodiments, the favorableintrogressed allele is selected from Sct_028: allele-3,Satt512:allele-2, S60211-TB:allele-1, Sat_117: allele-2, andP13158A:allele-2.

In other aspects, various software is used in determining linked markerloci. For example, TASSEL, MapManager-QTX, and GeneFlow all find usewith the invention. In some embodiments, such as when software is usedin the linkage analysis, the detected allele information (i.e., thedata) is electronically transmitted or electronically stored, forexample, in a computer readable medium.

In other aspects, various software are used in determining linked markerloci used to construct a transgenic plant. For example, TASSEL,MapManager-QTX, and GeneFlow all find use with the invention.

Systems for identifying a soybean plant predicted to have tolerance orimproved tolerance to Charcoal Rot Drought Complex are also a feature ofthe invention. Typically, the systems include a set of marker primersand/or probes configured to detect at least one favorable allele of oneor more marker locus associated with tolerance or improved tolerance toCharcoal Rot Drought Complex, wherein the marker locus or loci areselected from: Sct_028, Satt512, 560211-TB, Sat_117, 501954-1-A,P13158A, S63880-CB, 500415-1-A, S00705-1-A, and 502118-1-A as well asany other marker that is linked (or in some embodiments, closely linked,e.g., demonstrating not more than 10% recombination frequency) to theseQTL markers; and furthermore, any marker locus that is located withinthe chromosomal QTL intervals including:

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1));    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N).        In some embodiments, preferred QTL markers used are selected        from Satt512, SCT_028, S60211-TB, Sat_117, S01954-1-A, and,        S00415-1-A.

Where a system that performs marker detection or correlation is desired,the system can also include a detector that is configured to detect oneor more signal outputs from the set of marker probes or primers, oramplicon thereof, thereby identifying the presence or absence of theallele; and/or system instructions that correlate the presence orabsence of the favorable allele with the predicted tolerance. Theprecise configuration of the detector will depend on the type of labelused to detect the marker allele. Typical embodiments include lightdetectors, radioactivity detectors, and the like. Detection of the lightemission or other probe label is indicative of the presence or absenceof a marker allele. Similarly, the precise form of the instructions canvary depending on the components of the system, e.g., they can bepresent as system software in one or more integrated unit of the system,or can be present in one or more computers or computer readable mediaoperably coupled to the detector. In one typical embodiment, the systeminstructions include at least one look-up table that includes acorrelation between the presence or absence of the favorable allele andpredicted tolerance, improved tolerance or susceptibility.

In some embodiments, the system can be comprised of separate elements orcan be integrated into a single unit for convenient detection of markersalleles and for performing marker-tolerance trait correlations. In someembodiments, the system can also include a sample, for example, genomicDNA, amplified genomic DNA, cDNA, amplified cDNA, RNA, or amplified RNAfrom soybean or from a selected soybean plant tissue.

Kits are also a feature of the invention. For example, a kit can includeappropriate primers or probes for detecting tolerance associated markerloci and instructions in using the primers or probes for detecting themarker loci and correlating the loci with predicted Charcoal Rot DroughtComplex tolerance. The kits can further include packaging materials forpackaging the probes, primers or instructions, controls such as controlamplification reactions that include probes, primers or template nucleicacids for amplifications, molecular size markers, or the like.

Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular embodiments,which can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, terms in the singular and thesingular forms “a”, “an” and “the”, for example, include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “plant”, “the plant” or “a plant” also includes aplurality of plants; also, depending on the context, use of the term“plant” can also include genetically similar or identical progeny ofthat plant; use of the term “a nucleic acid” optionally includes, as apractical matter, many copies of that nucleic acid molecule; similarly,the term “probe” optionally (and typically) encompasses many similar oridentical probe molecules.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation. Numeric ranges recited within the specificationare inclusive of the numbers defining the range and include each integeror any non-integer fraction within the defined range. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the invention pertains. Although any methods and materials similaror equivalent to those described herein can be used in the practice fortesting of the present invention, the preferred materials and methodsare described herein. In describing and claiming the present invention,the following terminology will be used in accordance with thedefinitions set out below.

A “plant” can be a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to anyof: whole plants, plant components or organs (e.g., leaves, stems,roots, etc.), plant tissues, seeds, plant cells, and/or progeny of thesame. A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant. Thus, the term “soybeanplant” includes whole soybean plants, soybean plant cells, soybean plantprotoplast, soybean plant cell or soybean tissue culture from whichsoybean plants can be regenerated, soybean plant calli, soybean plantclumps and soybean plant cells that are intact in soybean plants orparts of soybean plants, such as soybean seeds, soybean pods, soybeanflowers, soybean cotyledons, soybean leaves, soybean stems, soybeanbuds, soybean roots, soybean root tips and the like.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells that can be cultured into a whole plant.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus. For example, a first allelecan occur on one chromosome, while a second allele occurs on a secondhomologous chromosome, e.g., as occurs for different chromosomes of aheterozygous individual, or between different homozygous or heterozygousindividuals in a population. A “favorable allele” is the allele at aparticular locus that confers, or contributes to, an agronomicallydesirable phenotype, e.g., tolerance to Charcoal Rot Drought Complex, oralternatively, is an allele that allows the identification ofsusceptible plants that can be removed from a breeding program orplanting. A favorable allele of a marker is a marker allele thatsegregates with the favorable phenotype, or alternatively, segregateswith susceptible plant phenotype, therefore providing the benefit ofidentifying disease-prone plants. A favorable allelic form of achromosome segment is a chromosome segment that includes a nucleotidesequence that contributes to superior agronomic performance at one ormore genetic loci physically located on the chromosome segment. “Allelefrequency” refers to the frequency (proportion or percentage) at whichan allele is present at a locus within an individual, within a line, orwithin a population of lines. For example, for an allele “A”, diploidindividuals of genotype “AA”, “Aa”, or “aa” have allele frequencies of1.0, 0.5, or 0.0, respectively. One can estimate the allele frequencywithin a line by averaging the allele frequencies of a sample ofindividuals from that line. Similarly, one can calculate the allelefrequency within a population of lines by averaging the allelefrequencies of lines that make up the population. For a population witha finite number of individuals or lines, an allele frequency can beexpressed as a count of individuals or lines (or any other specifiedgrouping) containing the allele.

An allele “positively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that the desired traitor trait form will occur in a plant comprising the allele. An allelenegatively correlates with a trait when it is linked to it and whenpresence of the allele is an indicator that a desired trait or traitform will not occur in a plant comprising the allele.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes). Anindividual is “heterozygous” if more than one allele type is present ata given locus (e.g., a diploid individual with one copy each of twodifferent alleles). The term “homogeneity” indicates that members of agroup have the same genotype at one or more specific loci. In contrast,the term “heterogeneity” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

A “locus” is a chromosomal region where a polymorphic nucleic acid,trait determinant, gene or marker is located. Thus, for example, a “genelocus” is a specific chromosome location in the genome of a specieswhere a specific gene can be found.

The term “quantitative trait locus” or “QTL” refers to a polymorphicgenetic locus with at least one allele that correlates with thedifferential expression of a phenotypic trait in at least one geneticbackground, e.g., in at least one breeding population or progeny. A QTLcan act through a single gene mechanism or by a polygenic mechanism.

The terms “marker”, “molecular marker”, “marker nucleic acid”, and“marker locus” refer to a nucleotide sequence or encoded product thereof(e.g., a protein) used as a point of reference when identifying a linkedlocus. A marker can be derived from genomic nucleotide sequence or fromexpressed nucleotide sequences (e.g., from a spliced RNA or a cDNA), orfrom an encoded polypeptide. The term also refers to nucleic acidsequences complementary to or flanking the marker sequences, such asnucleic acids used as probes or primer pairs capable of amplifying themarker sequence. A “marker probe” is a nucleic acid sequence or moleculethat can be used to identify the presence of a marker locus, e.g., anucleic acid probe that is complementary to a marker locus sequence.Alternatively, in some aspects, a marker probe refers to a probe of anytype that is able to distinguish (i.e., genotype) the particular allelethat is present at a marker locus. Nucleic acids are “complementary”when they specifically hybridize in solution, e.g., according toWatson-Crick base pairing rules. A “marker locus” is a locus that can beused to track the presence of a second linked locus, e.g., a linkedlocus that encodes or contributes to expression of a phenotypic trait.For example, a marker locus can be used to monitor segregation ofalleles at a locus, such as a QTL, that are genetically or physicallylinked to the marker locus. Thus, a “marker allele”, alternatively an“allele of a marker locus”, is one of a plurality of polymorphicnucleotide sequences found at a marker locus in a population that ispolymorphic for the marker locus. In some aspects, the present inventionprovides marker loci correlating with tolerance to Charcoal Rot DroughtComplex in soybean. Each of the identified markers is expected to be inclose physical and genetic proximity (resulting in physical and/orgenetic linkage) to a genetic element, e.g., a QTL that contributes totolerance.

“Genetic markers” are nucleic acids that are polymorphic in a populationand where the alleles of which can be detected and distinguished by oneor more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and thelike. The term also refers to nucleic acid sequences complementary tothe genomic sequences, such as nucleic acids used as probes.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by methods well-established in the art. Theseinclude, e.g., PCR-based sequence specific amplification methods,detection of restriction fragment length polymorphisms (RFLP), detectionof isozyme markers, detection of polynucleotide polymorphisms by allelespecific hybridization (ASH), detection of amplified variable sequencesof the plant genome, detection of self-sustained sequence replication,detection of simple sequence repeats (SSRs), detection of singlenucleotide polymorphisms (SNPs), or detection of amplified fragmentlength polymorphisms (AFLPs). Well established methods are also know forthe detection of expressed sequence tags (ESTs) and SSR markers derivedfrom EST sequences and randomly amplified polymorphic DNA (RAPD).

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form. “Geneticmapping” is the process of defining the linkage relationships of locithrough the use of genetic markers, populations segregating for themarkers, and standard genetic principles of recombination frequency. A“genetic map location” is a location on a genetic map relative tosurrounding genetic markers on the same linkage group where a specifiedmarker can be found within a given species. In contrast, a “physicalmap” of the genome refers to absolute distances (for example, measuredin base pairs or isolated and overlapping contiguous genetic fragments,e.g., contigs). A physical map of the genome does not take into accountthe genetic behavior (e.g., recombination frequencies) between differentpoints on the physical map.

A “genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis. A genetic recombination frequency can be expressed incentimorgans (cM), where one cM is the distance between two geneticmarkers that show a 1% recombination frequency (i.e., a crossing-overevent occurs between those two markers once in every 100 celldivisions).

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is “associated with” another marker locus or someother locus (for example, a tolerance locus).

As used herein, linkage equilibrium describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

As used herein, linkage disequilibrium describes a situation where twomarkers segregate in a non-random manner, i.e., have a recombinationfrequency of less than 50% (and by definition, are separated by lessthan 50 cM on the same linkage group). Markers that show linkagedisequilibrium are considered linked. Linkage occurs when the markerlocus and a linked locus are found together in progeny plants morefrequently than not together in the progeny plants. As used herein,linkage can be between two markers, or alternatively between a markerand a phenotype. A marker locus can be associated with (linked to) atrait, e.g., a marker locus can be associated with tolerance or improvedtolerance to a plant pathogen when the marker locus is in linkagedisequilibrium with the tolerance trait. The degree of linkage of amolecular marker to a phenotypic trait is measured, e.g., as astatistical probability of co-segregation of that molecular marker withthe phenotype.

As used herein, the linkage relationship between a molecular marker anda phenotype is given as a “probability” or “adjusted probability”. Theprobability value is the statistical likelihood that the particularcombination of a phenotype and the presence or absence of a particularmarker allele is random. Thus, the lower the probability score, thegreater the likelihood that a phenotype and a particular marker willco-segregate. In some aspects, the probability score is considered“significant” or “insignificant”. In some embodiments, a probabilityscore of 0.05 (p=0.05, or a 5% probability) of random assortment isconsidered a significant indication of co-segregation. However, thepresent invention is not limited to this particular standard, and anacceptable probability can be any probability of less than 50% (p=0.5).For example, a significant probability can be less than 0.25, less than0.20, less than 0.15, or less than 0.1.

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci or traits (or both). In either case, linkage disequilibriumimplies that the relevant loci are within sufficient physical proximityalong a length of a chromosome so that they segregate together withgreater than random (i.e., non-random) frequency (in the case ofco-segregating traits, the loci that underlie the traits are insufficient proximity to each other). Linked loci co-segregate more than50% of the time, e.g., from about 51% to about 100% of the time. Theterm “physically linked” is sometimes used to indicate that two loci,e.g., two marker loci, are physically present on the same chromosome.

Advantageously, the two linked loci are located in close proximity suchthat recombination between homologous chromosome pairs does not occurbetween the two loci during meiosis with high frequency, e.g., such thatlinked loci co-segregate at least about 90% of the time, e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 990.5%, 99.75%, or more of the time.

The phrase “closely linked”, in the present application, means thatrecombination between two linked loci occurs with a frequency of equalto or less than about 10% (i.e., are separated on a genetic map by notmore than 10 cM). Put another way, the closely linked loci co-segregateat least 90% of the time. Marker loci are especially useful in thepresent invention when they demonstrate a significant probability ofco-segregation (linkage) with a desired trait (e.g., pathogenictolerance). For example, in some aspects, these markers can be termedlinked QTL markers. In other aspects, especially useful molecularmarkers are those markers that are linked or closely linked.

In some aspects, linkage can be expressed as any desired limit or range.For example, in some embodiments, two linked loci are two loci that areseparated by less than 50 cM map units. In other embodiments, linkedloci are two loci that are separated by less than 40 cM. In otherembodiments, two linked loci are two loci that are separated by lessthan 30 cM. In other embodiments, two linked loci are two loci that areseparated by less than 25 cM. In other embodiments, two linked loci aretwo loci that are separated by less than 20 cM. In other embodiments,two linked loci are two loci that are separated by less than 15 cM. Insome aspects, it is advantageous to define a bracketed range of linkage,for example, between 10 and 20 cM, or between 10 and 30 cM, or between10 and 40 cM.

The more closely a marker is linked to a second locus, the better anindicator for the second locus that marker becomes. Thus, in oneembodiment, closely linked loci such as a marker locus and a secondlocus display an inter-locus recombination frequency of 10% or less,preferably about 9% or less, still more preferably about 8% or less, yetmore preferably about 7% or less, still more preferably about 6% orless, yet more preferably about 5% or less, still more preferably about4% or less, yet more preferably about 3% or less, and still morepreferably about 2% or less. In highly preferred embodiments, therelevant loci display a recombination a frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Two loci that are localized to thesame chromosome, and at such a distance that recombination between thetwo loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be“proximal to” each other. In some cases, two different markers can havethe same genetic map coordinates. In that case, the two markers are insuch close proximity to each other that recombination occurs betweenthem with such low frequency that it is undetectable.

When referring to the relationship between two genetic elements, such asa genetic element contributing to tolerance and a proximal marker,“coupling” phase linkage indicates the state where the “favorable”allele at the tolerance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest isphysically linked with an “unfavorable” allele at the proximal markerlocus, and the two “favorable” alleles are not inherited together (i.e.,the two loci are “out of phase” with each other).

As used herein, the terms “chromosome interval” or “chromosome segment”designate a contiguous linear span of genomic DNA that resides in plantaon a single chromosome. The genetic elements or genes located on asingle chromosome interval are physically linked. The size of achromosome interval is not particularly limited.

In some aspects, for example in the context of the present invention,generally the genetic elements located within a single chromosomeinterval are also genetically linked, typically within a geneticrecombination distance of, for example, less than or equal to 20 cM, oralternatively, less than or equal to 10 cM. That is, two geneticelements within a single chromosome interval undergo recombination at afrequency of less than or equal to 20% or 10%.

In one aspect, any marker of the invention is linked (genetically andphysically) to any other marker that is at or less than 50 cM distant.In another aspect, any marker of the invention is closely linked(genetically and physically) to any other marker that is in closeproximity, e.g., at or less than 10 cM distant. Two closely linkedmarkers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2,1, 0.75, 0.5 or 0.25 cM or less from each other.

The phrase “Charcoal Rot” refers to the plant disease caused by aninfection of the plant with the fungal pathogen Macrophomina phaseolina.While Charcoal Rot is more common in the presence of low-available watergrowth conditions, it can exist even in the absence of such growthconditions.

The phrase ‘Charcoal Rot Drought Complex’ or CRDC refers to a conditionin a plant in which the disease caused by an infection with the fungalpathogen Macrophomina phaseolina interacts with low-available watergrowth conditions to subdue the plant. It is a combination of theinfection of the fungus and the low-available water conditions that aremost commonly encountered under field conditions. Under these fieldconditions, the plant is stressed by both the pathogen and environmentand is subdued by the two stresses operating substantiallysimultaneously.

“Tolerance” or “improved tolerance” in a soybean plant to Charcoal RotDrought Complex is an indication that the soybean plant is less affectedwith respect to yield and/or survivability or other relevant agronomicmeasures, upon introduction of the causative agents of that disease,e.g., Macrophomina infection and low-available water growth conditions.“Tolerance” or “improved tolerance” in a soybean plant to Macrophominainfection is an indication that the soybean plant is less affected withrespect to yield and/or survivability or other relevant agronomicmeasures, upon infection of the plant with Macrophomina species, than aless tolerant or more “susceptible” plant. “Tolerance” or “improvedtolerance” in a soybean plant to low-available water growth conditionsis an indication that the soybean plant is less affected with respect toyield and/or survivability or other relevant agronomic measures, whenfaced with low-available water growth conditions or less-than-idealhydration conditions, than a less tolerant or more “susceptible” plant.Tolerance is a relative term, indicating that the infected plantproduces better yield of soybean than another, similarly treated, moresusceptible plant. That is, the conditions cause a reduced decrease insoybean survival and/or yield in a tolerant soybean plant, as comparedto a susceptible soybean plant.

One of skill will appreciate that soybean plant tolerance to CharcoalRot Drought Complex varies widely, can represent a spectrum of moretolerant or less tolerant phenotypes, and can vary depending on theseverity of the infection. However, by simple observation, one of skillcan determine the relative tolerance or susceptibility of differentplants, plant lines or plant families to Charcoal Rot Drought Complex,and furthermore, will also recognize the phenotypic gradations of“tolerant.”

Ratings are assigned by evaluating all plants of a cultivar in a 2 rowby 15 foot plot. Cultivar scores are based on a 1 to 9 system where ascore of ‘9’ would indicate that all plants in the plot are normal withno disease symptoms and a score of ‘1’ would indicate that all plants inthe plot are dead from disease. The experiments described herein scoresoybean tolerance to Charcoal Rot Drought Complex using the followingscale: 9=no disease symptoms with normal plant growth; 8=very slightsymptoms including up to a 10% reduction in leaflet and overall canopysize with no wilting; 7=wilting beginning to appear at the uppermost twonodes; 6=wilting at the uppermost three nodes and leaflet yellowingbeginning appear; 5=Up to 5% plant death with wilting and yellowing ofleaflets occurring at the uppermost four nodes; 4=Up to 10% plant deathwith wilting and yellowing of leaflets occurring at the uppermost fournodes; 3=Up to 25% plant death with wilting and yellowing of leafletsoccurring at the uppermost four nodes; 2=up to 50% plant death;1=50-100% plant death. FIG. 8 gives a representative example ofcultivars with vastly different Charcoal Rot Drought Complex toleranceusing this scoring system.

Charcoal Rot Drought Complex “tolerance” differs from Macrophomina“resistance” in that tolerance is a measure of a soybean plant's abilityto survive and yield soybean despite the presence of Macrophominainfection, as opposed to a measure of the soybean plant's ability toresist infection, just as low-available water growth condition tolerancedescribes a soybean plant's ability to survive and yield soybean despitethe existence of low-available water growth conditions. As used in theart, “tolerance” is sometimes referred to as “general resistance”,“rate-reducing resistance” or “partial resistance”.

The term “crossed” or “cross” in the context of this invention means thefusion of gametes via pollination to produce progeny (e.g., cells, seedsor plants). The term encompasses both sexual crosses (the pollination ofone plant by another) and selfing (self-pollination, e.g., when thepollen and ovule are from the same plant).

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g., aselected allele of a marker, a QTL, or the like. In any case, offspringcomprising the desired allele can be repeatedly backcrossed to a linehaving a desired genetic background and selected for the desired allele,to result in the allele becoming fixed in a selected genetic background.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendents that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor. Traditionally, a “subline” has been derived by inbreedingthe seed from an individual soybean plant selected at the F3 to F5generation until the residual segregating loci are “fixed” or homozygousacross most or all loci. Commercial soybean varieties (or lines) aretypically produced by aggregating (“bulking”) the self-pollinatedprogeny of a single F3 to F5 plant from a controlled cross between 2genetically different parents. While the variety typically appearsuniform, the self-pollinating variety derived from the selected planteventually (e.g., F8) becomes a mixture of homozygous plants that canvary in genotype at any locus that was heterozygous in the originallyselected F3 to F5 plant. In the context of the invention, marker-basedsublines, that differ from each other based on qualitative polymorphismat the DNA level at one or more specific marker loci, are derived bygenotyping a sample of seed derived from individual self-pollinatedprogeny derived from a selected F3-F5 plant. The seed sample can begenotyped directly as seed, or as plant tissue grown from such a seedsample. Optionally, seed sharing a common genotype at the specifiedlocus (or loci) are bulked providing a subline that is geneticallyhomogenous at identified loci important for a trait of interest (yield,tolerance, etc.).

An “ancestral line” is a parent line used as a source of genes e.g., forthe development of elite lines. An “ancestral population” is a group ofancestors that have contributed the bulk of the genetic variation thatwas used to develop elite lines. “Descendants” are the progeny ofancestors, and may be separated from their ancestors by many generationsof breeding. For example, elite lines are the descendants of theirancestors. A “pedigree structure” defines the relationship between adescendant and each ancestor that gave rise to that descendant. Apedigree structure can span one or more generations, describingrelationships between the descendant and its parents, grand parents,great-grand parents, etc.

An “elite line” or “elite strain” is an agronomically superior line thathas resulted from many cycles of breeding and selection for superioragronomic performance. Numerous elite lines are available and known tothose of skill in the art of soybean breeding. An “elite population” isan assortment of elite individuals or lines that can be used torepresent the state of the art in terms of agronomically superiorgenotypes of a given crop species, such as soybean. Similarly, an “elitegermplasm” or elite strain of germplasm is an agronomically superiorgermplasm, typically derived from and/or capable of giving rise to aplant with superior agronomic performance, such as an existing or newlydeveloped elite line of soybean.

In contrast, an “exotic soybean strain” or an “exotic soybean germplasm”is a strain or germplasm derived from a soybean not belonging to anavailable elite soybean line or strain of germplasm. In the context of across between two soybean plants or strains of germplasm, an exoticgermplasm is not closely related by descent to the elite germplasm withwhich it is crossed. Most commonly, the exotic germplasm is not derivedfrom any known elite line of soybean, but rather is selected tointroduce novel genetic elements (typically novel alleles) into abreeding program.

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods. An “amplicon” is an amplified nucleicacid, e.g., a nucleic acid that is produced by amplifying a templatenucleic acid by any available amplification method (e.g., PCR, LCR,transcription, or the like).

A “genomic nucleic acid” is a nucleic acid that corresponds in sequenceto a heritable nucleic acid in a cell. Common examples include nucleargenomic DNA and amplicons thereof. A genomic nucleic acid is, in somecases, different from a spliced RNA, or a corresponding cDNA, in thatthe spliced RNA or cDNA is processed, e.g., by the splicing machinery,to remove introns. Genomic nucleic acids optionally comprisenon-transcribed (e.g., chromosome structural sequences, promoterregions, or enhancer regions) and/or non-translated sequences (e.g.,introns), whereas spliced RNA/cDNA typically do not have non-transcribedsequences or introns. A “template nucleic acid” is a nucleic acid thatserves as a template in an amplification reaction (e.g., a polymerasebased amplification reaction such as PCR, a ligase mediatedamplification reaction such as LCR, a transcription reaction, or thelike). A template nucleic acid can be genomic in origin, oralternatively, can be derived from expressed sequences, e.g., a cDNA oran EST.

An “exogenous nucleic acid” is a nucleic acid that is not native to aspecified system (e.g., a germplasm, plant, or variety), with respect tosequence, genomic position, or both. As used herein, the terms“exogenous” or “heterologous” as applied to polynucleotides orpolypeptides typically refers to molecules that have been artificiallysupplied to a biological system (e.g., a plant cell, a plant gene, aparticular plant species or variety or a plant chromosome under study)and are not native to that particular biological system. The terms canindicate that the relevant material originated from a source other thana naturally occurring source, or can refer to molecules having anon-natural configuration, genetic location or arrangement of parts.

In contrast, for example, a “native” or “endogenous” gene is a gene thatdoes not contain nucleic acid elements encoded by sources other than thechromosome or other genetic element on which it is normally found innature. An endogenous gene, transcript or polypeptide is encoded by itsnatural chromosomal locus, and not artificially supplied to the cell.

The term “recombinant” in reference to a nucleic acid or polypeptideindicates that the material (e.g., a recombinant nucleic acid, gene,polynucleotide, or polypeptide) has been altered by human intervention.Generally, the arrangement of parts of a recombinant molecule is not anative configuration, or the primary sequence of the recombinantpolynucleotide or polypeptide has in some way been manipulated. Thealteration to yield the recombinant material can be performed on thematerial within or removed from its natural environment or state. Forexample, a naturally occurring nucleic acid becomes a recombinantnucleic acid if it is altered, or if it is transcribed from DNA whichhas been altered, by means of human intervention performed within thecell from which it originates. A gene sequence open reading frame isrecombinant if that nucleotide sequence has been removed from it naturalcontext and cloned into any type of artificial nucleic acid vector.Protocols and reagents to produce recombinant molecules, especiallyrecombinant nucleic acids, are common and routine in the art. In oneembodiment, an artificial chromosome can be created and inserted intomaize plants by any method known in the art (e.g., direct transferprocesses, such as, e.g., PEG-induced DNA uptake, protoplast fusion,microinjection, electroporation, and microprojectile bombardment). Anartificial chromosome is a piece of DNA that can stably replicate andsegregate alongside endogenous chromosomes. It has the capacity toaccommodate and express heterologous genes inserted therein. Integrationof heterologous DNA into the megareplicator region (primary replicationinitiation site of centromeres) or in close proximity thereto, initiatesa large-scale amplification of megabase-size chromosomal segments, whichleads to de novo chromosome formation. See, e.g., U.S. Pat. No.6,077,697, incorporated herein by reference.

The term recombinant can also refer to an organism that harborsrecombinant material, e.g., a plant that comprises a recombinant nucleicacid is considered a recombinant plant. In some embodiments, arecombinant organism is a transgenic organism.

The term “introduced” when referring to translocating a heterologous orexogenous nucleic acid into a cell refers to the incorporation of thenucleic acid into the cell using any methodology. The term encompassessuch nucleic acid introduction methods as “transfection”,“transformation” and “transduction”.

As used herein, the term “vector” is used in reference to polynucleotideor other molecules that transfer nucleic acid segment(s) into a cell.The term “vehicle” is sometimes used interchangeably with “vector”. Avector optionally comprises parts which mediate vector maintenance andenable its intended use (e.g., sequences necessary for replication,genes imparting drug or antibiotic resistance, a multiple cloning site,or operably linked promoter/enhancer elements which enable theexpression of a cloned gene). Vectors are often derived from plasmids,bacteriophages, or plant or animal viruses. A “cloning vector” or“shuttle vector” or “subcloning vector” contains operably linked partsthat facilitate subcloning steps (e.g., a multiple cloning sitecontaining multiple restriction endonuclease sites).

The term “expression vector” as used herein refers to a vectorcomprising operably linked polynucleotide sequences that facilitateexpression of a coding sequence in a particular host organism (e.g., abacterial expression vector or a plant expression vector).Polynucleotide sequences that facilitate expression in prokaryotestypically include, e.g., a promoter, an operator (optional), and aribosome binding site, often along with other sequences. Eukaryoticcells can use promoters, enhancers, termination and polyadenylationsignals and other sequences that are generally different from those usedby prokaryotes.

The term “transgenic plant” refers to a plant that comprises within itscells a heterologous polynucleotide. Generally, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette. “Transgenic” is used herein to refer toany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenic organisms or cells initially so altered, aswell as those created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (e.g.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

A specified nucleic acid is “derived from” a given nucleic acid when itis constructed using the given nucleic acid's sequence, or when thespecified nucleic acid is constructed using the given nucleic acid. Forexample, a cDNA or EST is derived from an expressed mRNA.

The term “genetic element” or “gene” refers to a heritable sequence ofDNA, i.e., a genomic sequence, with functional significance. The term“gene” can also be used to refer to, e.g., a cDNA and/or a mRNA encodedby a genomic sequence, as well as to that genomic sequence.

The term “genotype” is the genetic constitution of an individual (orgroup of individuals) at one or more genetic loci, as contrasted withthe observable trait (the phenotype). Genotype is defined by theallele(s) of one or more known loci that the individual has inheritedfrom its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple loci,or, more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome. A“haplotype” is the genotype of an individual at a plurality of geneticloci. Typically, the genetic loci described by a haplotype arephysically and genetically linked, i.e., on the same chromosome segment.

The terms “phenotype”, or “phenotypic trait” or “trait” refers to one ormore trait of an organism. The phenotype can be observable to the nakedeye, or by any other means of evaluation known in the art, e.g.,microscopy, biochemical analysis, genomic analysis, or an assay for aparticular disease resistance. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, i.e., a “single genetrait”. In other cases, a phenotype is the result of several genes.

A “molecular phenotype” is a phenotype detectable at the level of apopulation of (one or more) molecules. Such molecules can be nucleicacids such as genomic DNA or RNA, proteins, or metabolites. For example,a molecular phenotype can be an expression profile for one or more geneproducts, e.g., at a specific stage of plant development, in response toan environmental condition or stress, etc. Expression profiles aretypically evaluated at the level of RNA or protein, e.g., on a nucleicacid array or “chip” or using antibodies or other binding proteins.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. For example, yield ofsoybean is commonly measured in bushels of seed per acre or metric tonsof seed per hectare per season. Yield is affected by both genetic andenvironmental factors. “Agronomics”, “agronomic traits”, and “agronomicperformance” refer to the traits (and underlying genetic elements) of agiven plant variety that contribute to yield over the course of growingseason. Individual agronomic traits include emergence vigor, vegetativevigor, stress tolerance, disease resistance or tolerance, herbicideresistance, branching, flowering, seed set, seed size, seed density,standability, threshability and the like. Yield is, therefore, the finalculmination of all agronomic traits.

A “set” of markers or probes refers to a collection or group of markersor probes, or the data derived therefrom, used for a common purpose,e.g., identifying soybean plants with a desired trait (e.g., toleranceto Charcoal Rot Drought Complex). Frequently, data corresponding to themarkers or probes, or data derived from their use, is stored in anelectronic medium. While each of the members of a set possess utilitywith respect to the specified purpose, individual markers selected fromthe set as well as subsets including some, but not all of the markers,are also effective in achieving the specified purpose.

A “look up table” is a table that correlates one form of data toanother, or one or more forms of data with a predicted outcome that thedata is relevant to. For example, a look up table can include acorrelation between allele data and a predicted trait that a plantcomprising a given allele is likely to display. These tables can be, andtypically are, multidimensional, e.g., taking multiple alleles intoaccount simultaneously, and, optionally, taking other factors intoaccount as well, such as genetic background, e.g., in making a traitprediction.

A “computer readable medium” is an information storage media that can beaccessed by a computer using an available or custom interface. Examplesinclude memory (e.g., ROM, RAM, or flash memory), optical storage media(e.g., CD-ROM), magnetic storage media (computer hard drives, floppydisks, etc.), punch cards, and many others that are commerciallyavailable. Information can be transmitted between a system of interestand the computer, or to or from the computer to or from the computerreadable medium for storage or access of stored information. Thistransmission can be an electrical transmission, or can be made by otheravailable methods, such as an IR link, a wireless connection, or thelike.

“System instructions” are instruction sets that can be partially orfully executed by the system. Typically, the instruction sets arepresent as system software.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

FIG. 1 provides a table listing soybean markers demonstrating linkagedisequilibrium with the Charcoal Rot Drought Complex tolerance phenotypeas determined by intergroup allele frequency distribution analysis,association mapping analysis, QTL interval mapping (including singlemarker regression analysis), and marker regression and interval mappinganalysis using MapManager. The table indicates the genomic-SSR orEST-SSR marker type (all simple sequence repeats) or SNP markers, thechromosome on which the marker is located and its approximate geneticmap position relative to other known markers, given in cM, with positionzero being the first (most distal) marker on the chromosome, as providedin the integrated genetic map in FIG. 6. Also shown are the soybeanpopulations used in the analysis and the statistical probability ofrandom segregation of the marker and the tolerance/susceptibilityphenotype given as an adjusted probability taking into account thevariability and false positives of multiple tests. Probability valuesfrom single marker regression are also shown.

FIG. 2 provides a table listing genomic and EST SSR markers, includingthose markers that demonstrated linkage disequilibrium with the CharcoalRot Drought Complex tolerance phenotype. The table provides thesequences of the left and right PCR primers used in the SSR marker locusgenotyping analysis. Also shown is the pigtail sequence used on the 5′end of the right primer, and the number of nucleotides in the tandemrepeating element in the SSR.

FIG. 3 provides a table listing the SNP markers that demonstratedlinkage disequilibrium with the Charcoal Rot Drought Complex tolerancephenotype. The table provides the sequences of the PCR primers used togenerate a SNP-containing amplicon, and the allele-specific probes thatwere used to identify the SNP allele in an allele-specific hybridizationassay (ASH assay).

FIG. 4 provides an allele dictionary for the alleles of the SSR markersshown in FIG. 1, including those markers that demonstrated linkagedisequilibrium with the Charcoal Rot Drought Complex tolerancephenotype. Each allele is defined by the size of a PCR amplicongenerated from soybean genomic DNA or mRNA using the primers listed inFIG. 2. Sizes of the PCR amplicons are indicated in base pairs (bp).

FIG. 5 provides a table listing genetic markers that are linked andgenetic markers that are closely linked to the Charcoal Rot DroughtComplex tolerance markers identified by the present invention.

FIG. 6 (6.1-6.21) provides an integrated genetic map of soybean markers.These markers are distributed over each soybean chromosome. The geneticmap positions of the markers are indicated in centiMorgans (cM),typically with position zero being the first (most distal) marker on thechromosome. The markers within the linked interval, and closely linkedinterval around a central marker are indicated.

FIG. 7 provides a table listing the soybean lines used in the currentCharcoal Rot Drought Complex tolerance analysis, the Charcoal RotDrought Complex tolerance score of each line used, and whether theparticular line was used in the Trait Allele and/or InterGroup analyses.

FIG. 8 provides an example of cultivars with vastly different CharcoalRot Drought Complex tolerance scores. Using the scoring system describedherein, the two row plot of one cultivar of soybean plants on the leftscored a 6 while the two row plot of a different cultivar of soybeanplants on the right scored a 1. This was based on the determination thatthe two row plot of one cultivar of soybean plants on the left hadwilting at the uppermost three nodes and leaflet yellowing beginningappear, while the two row plot of a different cultivar of soybean plantson the right had near 100% plant death.

DETAILED DESCRIPTION OF THE INVENTION

Charcoal Rot is a disease of soybean, causing reduced plant viabilityand reductions in yield. This disease is caused by infection of theplant with Macrophomina phaseolina, a fungal pathogen. Though thisdisease is most prevalent during low-available water growth conditions,it can exist even in the absence of such growth conditions. WhileMacrophomina resistant plants have been previously developed, the strongselective pressures that resistant soybean impose on Macrophomina islikely to cause relatively rapid loss of resistance against races ofMacrophomina that evolve to combat resistance traits in the resistantsoybean, as has been seen with other soybean fungal pathogens, such asSclerotinia. Accordingly, tolerance to Charcoal Rot and/or Macrophominainfection, in which the plant survives, thrives and produces highyields, despite a productive Macrophomina infection, is an alternatestrategy to combat losses due to Charcoal Rot and/or Macrophominainfection. That is, there is not a strong negative selection againstMacrophomina imposed by tolerance, because tolerant soybean plantssupport a productive Macrophomina infection.

Further, as plant stress caused by low-available water growth conditionsis related to the existence and severity of Charcoal Rot and/orMacrophomina infection, with plants showing reduced survivability andyield from these conditions when coupled with low-available water growthconditions, soybean plants tolerant to low-available water growthconditions would show increased Charcoal Rot and/or Macrophominainfection tolerance, as well, and are therefore desirable. In addition,as low-available water growth condition is itself a major cause of lossof plant viability and yield, even in the absence of Charcoal Rot and/orMacrophomina infection, plants tolerant to such growth conditions aredesirable for their direct benefits, not related to Charcoal Rot aswell.

The identification and selection of soybean plants that show toleranceto Charcoal Rot Drought Complex using MAS can provide an effective andenvironmentally friendly approach to overcoming losses caused by thisdisease. The present invention provides soybean marker loci thatdemonstrate statistically significant co-segregation with Charcoal RotDrought Complex tolerance. Detection of these loci or additional linkedloci can be used in marker assisted soybean breeding programs to producetolerant plants, or plants with improved tolerance. The linked SSR andSNP markers identified herein are provided in FIG. 1. These markersinclude Sct_028, Satt512, S60211-TB, Sat_117, S01954-1-A, P13158A,S63880-CB, S00415-1-A, S00705-1-A, and S02118-1-A.

Each of the SSR-type markers display a plurality of alleles that can bevisualized as different sized PCR amplicons, as summarized in the SSRallele dictionary in FIG. 4. The PCR primers that are used to generatethe SSR-marker amplicons are provided in FIG. 2. The alleles of SNP-typemarkers are determined using an allele-specific hybridization protocol,as known in the art. The PCR primers used to amplify the SNP domain, andthe allele-specific probes used to genotype the locus are provided inFIG. 3.

As recognized in the art, any other marker that is linked to a QTLmarker (e.g., a disease tolerance marker) also finds use for that samepurpose. Examples of additional markers that are linked to the diseasetolerance markers recited herein are provided. For example, a linkedmarker can be determined from the soybean consensus genetic map providedin FIG. 6. Additional linked and closely linked markers are furtherprovided in FIG. 5. It is not intended, however, that linked markersfinding use with the invention be limited to those recited in FIG. 5 or6.

The invention also provides chromosomal QTL intervals that correlatewith Charcoal Rot Drought Complex tolerance. These intervals are locatedon linkage groups C2, E, B2, G, H, B1, C1, D1b and N. Any marker locatedwithin these intervals finds use as a marker for Charcoal Rot DroughtComplex tolerance. These intervals include:

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1);    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N).

Methods for identifying soybean plants or germplasm that carry preferredalleles of tolerance marker loci are a feature of the invention. Inthese methods, any of a variety of marker detection protocols is used toidentify marker loci, depending on the type of marker loci. Typicalmethods for marker detection include amplification and detection of theresulting amplified markers, e.g., by PCR, LCR, transcription basedamplification methods, or the like. These include ASH, SSR detection,RFLP analysis and many others.

Although particular marker alleles can show co-segregation with adisease tolerance or susceptibility phenotype, it is important to notethat the marker locus is not necessarily part of the QTL locusresponsible for the tolerance or susceptibility. For example, it is nota requirement that the marker polynucleotide sequence be part of a genethat imparts disease resistance (for example, be part of the gene openreading frame). The association between a specific marker allele withthe tolerance or susceptibility phenotype is due to the original“coupling” linkage phase between the marker allele and the QTL toleranceor susceptibility allele in the ancestral soybean line from which thetolerance or susceptibility allele originated. Eventually, with repeatedrecombination, crossing over events between the marker and QTL locus canchange this orientation. For this reason, the favorable marker allelemay change depending on the linkage phase that exists within thetolerant parent used to create segregating populations. This does notchange the fact that the genetic marker can be used to monitorsegregation of the phenotype. It only changes which marker allele isconsidered favorable in a given segregating population.

Identification of soybean plants or germplasm that include a markerlocus or marker loci linked to a tolerance trait or traits provides abasis for performing marker assisted selection of soybean. Soybeanplants that comprise favorable markers or favorable alleles are selectedfor, while soybean plants that comprise markers or alleles that arenegatively correlated with tolerance can be selected against. Desiredmarkers and/or alleles can be introgressed into soybean having a desired(e.g., elite or exotic) genetic background to produce an introgressedtolerant soybean plant or germplasm. In some aspects, it is contemplatedthat a plurality of tolerance markers are sequentially or simultaneousselected and/or introgressed. The combinations of tolerance markers thatare selected for in a single plant is not limited, and can include anycombination of markers recited in FIG. 1, any markers linked to themarkers recited in FIG. 1, or any markers located within the QTLintervals defined herein.

Various methods are known in the art for determining (and measuring) thetolerance of a soybean plant to Charcoal Rot Drought Complex. Theydescribe a tolerance measurement scale of 1-9, with 9=no disease and1=total necrosis caused by Macrophomina phaseolina. It will beappreciated that all such scales are relative and that numbering andprecise correlation to any scale can be performed at the discretion ofthe practitioner.

Typically, individual field tests are monitored for Charcoal Rotsymptoms during the middle to late vegetative stages, but such symptomstypically appear in the early reproductive stage (during flowering andearly pod set). Data collection is usually done in 3 or 4 successivescorings about 7 days apart. Scorings continue until worsening symptomscan no longer be quantified or until the symptoms are confounded byother factors such as other diseases, insect pressure, severe weather,or advancing maturity.

In general, while there is a certain amount of subjectivity to assigningseverity measurements for disease caused symptoms, assignment to a givenscale as noted above is well within the skill of a practitioner in thefield. Measurements can also be averaged across multiple scorers toreduce variation in field measurements. Furthermore, although protocolsusing artificial inoculation of field nurseries with Macrophominaphaseolina can certainly be used in assessing tolerance, it is alsotypical for tolerance ratings to be based on actual field observationsof fortuitous natural disease incidence, with the informationcorresponding to disease incidence for a cultivar being averaged overmany locations and, typically, several years of crop growing.

If there is no disease present, the rating system above is inapplicable,because everything in an uninfected field scores as tolerant. However,if Charcoal Rot does occur in a specific field location, all of thelines at that location can be scored as noted above. These scores canaccumulate over locations and years to show disease tolerance for givencultivars. Thus, older lines can have more years of observation thannewer ones etc. However, relative measurements can easily be made usingthe scoring system noted above. Furthermore, the tolerance ratings canbe updated and refined each year based on the previous year'sobservations in the field. Based on this, Charcoal Rot scores for acultivar are relative measurements of tolerance.

The experiments described herein score soybean tolerance to Charcoal RotDrought Complex using the following scale: 9=no disease symptoms withnormal plant growth; 8=very slight symptoms including up to a 10%reduction in leaflet and overall canopy size with no wilting; 7=wiltingbeginning to appear at the uppermost two nodes; 6=wilting at theuppermost three nodes and leaflet yellowing beginning appear; 5=Up to 5%plant death with wilting and yellowing of leaflets occurring at theuppermost four nodes; 4=Up to 10% plant death with wilting and yellowingof leaflets occurring at the uppermost four nodes; 3=Up to 25% plantdeath with wilting and yellowing of leaflets occurring at the uppermostfour nodes; 2=up to 50% plant death; 1=50-100% plant death. FIG. 8 givesa representative example of cultivars with vastly different Charcoal RotDrought Complex tolerance using this scoring system.

Tolerance assays are useful to verify that the tolerance trait stillsegregates with the marker in any particular plant or population, and,of course, to measure the degree of tolerance improvement achieved byintrogressing or recombinantly introducing the trait into a desiredbackground.

Systems, including automated systems for selecting plants that comprisea marker of interest and/or for correlating presence of the marker withtolerance are also a feature of the invention. These systems can includeprobes relevant to marker locus detection, detectors for detectinglabels on the probes, appropriate fluid handling elements andtemperature controllers that mix probes and templates and/or amplifytemplates, and systems instructions that correlate label detection tothe presence of a particular marker locus or allele.

Kits are also a feature of the invention. For example, a kit can includeappropriate primers or probes for detecting tolerance associated markerloci and instructions in using the primers or probes for detecting themarker loci and correlating the loci with predicted Charcoal Rot DroughtComplex tolerance. The kits can further include packaging materials forpackaging the probes, primers or instructions, controls such as controlamplification reactions that include probes, primers or template nucleicacids for amplifications, molecular size markers, or the like.

Tolerance Markers and Favorable Alleles

In traditional linkage analysis, no direct knowledge of the physicalrelationship of genes on a chromosome is required. Mendel's first law isthat factors of pairs of characters are segregated, meaning that allelesof a diploid trait separate into two gametes and then into differentoffspring. Classical linkage analysis can be thought of as a statisticaldescription of the relative frequencies of cosegregation of differenttraits. Linkage analysis is the well characterized descriptive frameworkof how traits are grouped together based upon the frequency with whichthey segregate together. That is, if two non-allelic traits areinherited together with a greater than random frequency, they are saidto be “linked”. The frequency with which the traits are inheritedtogether is the primary measure of how tightly the traits are linked,i.e., traits which are inherited together with a higher frequency aremore closely linked than traits which are inherited together with lower(but still above random) frequency. Traits are linked because the geneswhich underlie the traits reside on the same chromosome. The furtherapart on a chromosome the genes reside, the less likely they are tosegregate together, because homologous chromosomes recombine duringmeiosis. Thus, the further apart on a chromosome the genes reside, themore likely it is that there will be a crossing over event duringmeiosis that will result in two genes segregating separately intoprogeny.

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or, also commonly, in centiMorgans (cM). ThecM is named after the pioneering geneticist Thomas Hunt Morgan and is aunit of measure of genetic recombination frequency. One cM is equal to a1% chance that a trait at one genetic locus will be separated from atrait at another locus due to crossing over in a single generation(meaning the traits segregate together 99% of the time). Becausechromosomal distance is approximately proportional to the frequency ofcrossing over events between traits, there is an approximate physicaldistance that correlates with recombination frequency. For example, insoybean, 1 cM correlates, on average, to about 400,000 base pairs (400Kb).

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, in the context of the present invention, one cM isequal to a 1% chance that a marker locus will be separated from anotherlocus (which can be any other trait, e.g., another marker locus, oranother trait locus that encodes a QTL), due to crossing over in asingle generation. The markers herein, as described in FIG. 1, e.g.,Sct_028, Satt512, S60211-TB, Sat_117, 501954-1-A, P13158A, 563880-CB,S00415-1-A, S00705-1-A, S02118-1-A as well as any of the chromosomeintervals:

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1);    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N)        have been found to correlate with tolerance, improved tolerance        or susceptibility to Charcoal Rot Drought Complex in soybean.        This means that the markers are sufficiently proximal to a        tolerance trait that they can be used as a predictor for the        tolerance trait. This is extremely useful in the context of        marker assisted selection (MAS), discussed in more detail        herein. In brief, soybean plants or germplasm can be selected        for markers or marker alleles that positively correlate with        tolerance, without actually raising soybean and measuring for        tolerance or improved tolerance (or, contrarily, soybean plants        can be selected against if they possess markers that negatively        correlate with tolerance or improved tolerance). MAS is a        powerful shortcut to selecting for desired phenotypes and for        introgressing desired traits into cultivars of soybean (e.g.,        introgressing desired traits into elite lines). MAS is easily        adapted to high throughput molecular analysis methods that can        quickly screen large numbers of plant or germplasm genetic        material for the markers of interest and is much more cost        effective than raising and observing plants for visible traits.

In some embodiments, the most preferred QTL markers are a subset of themarkers provided in FIG. 1. For example, the most preferred markers areSatt512, SCT_028, S60211-TB, Sat_177, S01954-1-A, and S00415-1-A.

When referring to the relationship between two genetic elements, such asa genetic element contributing to tolerance and a proximal marker,“coupling” phase linkage indicates the state where the “favorable”allele at the tolerance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest (e.g., aQTL for tolerance) is physically linked with an “unfavorable” allele atthe proximal marker locus, and the two “favorable” alleles are notinherited together (i.e., the two loci are “out of phase” with eachother).

A favorable allele of a marker is that allele of the marker thatco-segregates with a desired phenotype (e.g., disease tolerance). Asused herein, a QTL marker has a minimum of one favorable allele,although it is possible that the marker might have two or more favorablealleles found in the population. Any favorable allele of that marker canbe used advantageously for the identification and construction oftolerant soybean lines. Optionally, one, two, three or more favorableallele(s) of different markers are identified in, or introgressed into aplant, and can be selected for or against during MAS. Desirably, plantsor germplasm are identified that have at least one such favorable allelethat positively correlates with tolerance or improved tolerance.

Alternatively, a marker allele that co-segregates with diseasesusceptibility also finds use with the invention, since that allele canbe used to identify and counter select disease-susceptible plants. Suchan allele can be used for exclusionary purposes during breeding toidentify alleles that negatively correlate with tolerance, to eliminatesusceptible plants or germplasm from subsequent rounds of breeding.

In some embodiments of the invention, a plurality of marker alleles aresimultaneously selected for in a single plant or a population of plants.In these methods, plants are selected that contain favorable allelesfrom more than one tolerance marker, or alternatively, favorable allelesfrom more than one tolerance marker are introgressed into a desiredsoybean germplasm. One of skill in the art recognizes that thesimultaneous selection of favorable alleles from more than one diseasetolerance marker in the same plant is likely to result in an additive(or even synergistic) protective effect for the plant.

One of skill recognizes that the identification of favorable markeralleles is germplasm-specific. The determination of which marker allelescorrelate with tolerance (or susceptibility) is determined for theparticular germplasm under study. One of skill recognizes that methodsfor identifying the favorable alleles are routine and well known in theart, and furthermore, that the identification and use of such favorablealleles is well within the scope of the invention. Furthermore still,identification of favorable marker alleles in soybean populations otherthan the populations used or described herein is well within the scopeof the invention.

Amplification primers for amplifying SSR-type marker loci are a featureof the invention. Another feature of the invention is primers specificfor the amplification of SNP domains (SNP markers), and the probes thatare used to genotype the SNP sequences. FIGS. 2 and 3 provide specificprimers for marker locus amplification and probes for detectingamplified marker loci. However, one of skill will immediately recognizethat other sequences to either side of the given primers can be used inplace of the given primers, so long as the primers can amplify a regionthat includes the allele to be detected. Further, it will be appreciatedthat the precise probe to be used for detection can vary, e.g., anyprobe that can identify the region of a marker amplicon to be detectedcan be substituted for those examples provided herein. Further, theconfiguration of the amplification primers and detection probes can, ofcourse, vary. Thus, the invention is not limited to the primers andprobes specifically recited herein.

In some aspects, methods of the invention utilize an amplification stepto detect/genotype a marker locus. However, it will be appreciated thatamplification is not a requirement for marker detection—for example, onecan directly detect unamplified genomic DNA simply by performing aSouthern blot on a sample of genomic DNA. Procedures for performingSouthern blotting, amplification (PCR, LCR, or the like) and many othernucleic acid detection methods are well established and are taught,e.g., in Sambrook, et al., (2000) Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (“Sambrook”); Current Protocols in Molecular Biology, Ausubel, etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2002) (“Ausubel”)) and PCR Protocols A Guide to Methods and Applications(Innis, et al., eds) Academic Press Inc. San Diego, Calif. (1990)(Innis). Additional details regarding detection of nucleic acids inplants can also be found, e.g., in Plant Molecular Biology (1993) Croy(ed.) BIOS Scientific Publishers, Inc.

Separate detection probes can also be omitted in amplification/detectionmethods, e.g., by performing a real time amplification reaction thatdetects product formation by modification of the relevant amplificationprimer upon incorporation into a product, incorporation of labelednucleotides into an amplicon, or by monitoring changes in molecularrotation properties of amplicons as compared to unamplified precursors(e.g., by fluorescence polarization).

Typically, molecular markers are detected by any established methodavailable in the art, including, without limitation, allele specifichybridization (ASH) or other methods for detecting single nucleotidepolymorphisms (SNP), amplified fragment length polymorphism (AFLP)detection, amplified variable sequence detection, randomly amplifiedpolymorphic DNA (RAPD) detection, restriction fragment lengthpolymorphism (RFLP) detection, self-sustained sequence replicationdetection, simple sequence repeat (SSR) detection, single-strandconformation polymorphisms (SSCP) detection, isozyme markers detection,or the like. Any of the aforementioned marker types can be employed inthe context of the invention to identify chromosome segmentsencompassing genetic element that contribute to superior agronomicperformance (e.g., tolerance or improved tolerance).

QTL Chromosome Intervals

In some aspects, the invention provides QTL chromosome intervals, wherea QTL (or multiple QTLs) that segregate with Charcoal Rot DroughtComplex tolerance are contained in those intervals. A variety of methodswell known in the art are available for identifying chromosome intervals(also as described in detail in Example 1). The boundaries of suchchromosome intervals are drawn to encompass markers that will be linkedto one or more QTL. In other words, the chromosome interval is drawnsuch that any marker that lies within that interval (including theterminal markers that define the boundaries of the interval) can be usedas markers for disease tolerance. Each interval comprises at least oneQTL, and furthermore, may indeed comprise more than one QTL. Closeproximity of multiple QTL in the same interval may obfuscate thecorrelation of a particular marker with a particular QTL, as one markermay demonstrate linkage to more than one QTL. Conversely, e.g., if twomarkers in close proximity show co-segregation with the desiredphenotypic trait, it is sometimes unclear if each of those markersidentifying the same QTL or two different QTL. Regardless, knowledge ofhow many QTL are in a particular interval is not necessary to make orpractice the invention.

The present invention provides soybean chromosome intervals, where themarkers within that interval demonstrate co-segregation with toleranceto Charcoal Rot Drought Complex. Thus, each of these intervals comprisesat least one Charcoal Rot Drought Complex tolerance QTL. These intervalsare:

Linkage Method(s) of Group Flanking Markers Identification C2 Satt286and Satt371 Trait Allele Correlation and Intergroup Analysis E Satt575and Sat_136 Trait Allele Correlation and Intergroup Analysis B2 Satt467and Satt416 Intergroup Analysis G Sat_158 and A681_1 Trait AlleleCorrelation and Intergroup Analysis H Satt444 and A162_1 Trait AlleleCorrelation B1 Satt444 and Sat_331 Trait Allele Correlation C1 Bng019_1and Sct_191 Marker Regression and Interval Mapping Analysis D1b A605_1and A519_2 Marker Regression and Interval Mapping Analysis N Sat_306 andA363_3 Marker Regression and Interval Mapping Analysis

Each of the intervals described above shows a clustering of markers thatco-segregate with Charcoal Rot Drought Complex tolerance. Thisclustering of markers occurs in relatively small domains on the linkagegroups, indicating the presence of one or more QTL in those chromosomeregions. QTL intervals were drawn to encompass the markers thatco-segregate with tolerance. The intervals are defined by the markers ontheir termini, where the interval encompasses all the markers that mapwithin the interval as well as the markers that define the termini.

In some cases, an interval can be drawn, where the interval is definedby linkage to a preferred marker. For example, an interval on LG-C2 isdefined where any marker that is linked to the marker Sct_028 is amember of that interval. For example, as used here, linkage is definedas any marker that is within 25 cM from Sct_028. This interval on LG-C2is further illustrated in FIG. 5. The experimentally demonstrated markerSct_028 is shown, as are markers that are linked to Sct_028 (e.g.,within 25 cM of Sct_028) as determined by any suitable genetic linkagemap (for example, the GmComposite 2003 map found on the Soybasewebsite). These markers are shown in genetic order. Each of the markerslisted, including the terminal markers Satt286 and Satt371, are membersof the interval. The Satt286 and Satt371 markers are known in the art.

As described above, an interval (e.g., a chromosome interval or a QTLinterval) need not depend on an absolute measure of interval size suchas a centimorgans value. An interval can be described by the terminalmarkers that define the endpoints of the interval, and typically theinterval will include the terminal markers that define the extent of theinterval. An interval can include any marker localizing within thatchromosome domain, whether those markers are currently known or unknown.In situations where the interval is close to or comprises one end of thelinkage group, the interval can be described by one marker, for examplethe interval on linkage group G can be described as including markerSatt612 and below, the interval on linkage group N can be described asincluding marker Sat_306 and below, and the interval on linkage group Ecan be described as including marker Sat_136 and above, where “above”and “below” are the terms commonly used in the art to describe themarker's position relative to the distal end (position zero), with abovebeing closer to position zero. The invention provides a variety of meansfor defining a chromosome interval, for example, the marker lociprovided in the genetic map in FIG. 6, in the lists of linked markers ofFIG. 5, and in references cited herein (e.g., Song et al., (2004) “A NewIntegrated Genetic Linkage Map of the Soybean” Theor Appl Genet109:122-128).

Genetic Maps

As one of skill in the art will recognize, recombination frequencies(and as a result, genetic map positions) in any particular populationare not static. The genetic distances separating two markers (or amarker and a QTL) can vary depending on how the map positions aredetermined. For example, variables such as the parental mappingpopulations used, the software used in the marker mapping or QTLmapping, and the parameters input by the user of the mapping softwarecan contribute to the QTL/marker genetic map relationships. However, itis not intended that the invention be limited to any particular mappingpopulations, use of any particular software, or any particular set ofsoftware parameters to determine linkage of a particular marker orchromosome interval with the Charcoal Rot Drought Complex tolerancephenotype. It is well within the ability of one of ordinary skill in theart to extrapolate the novel features described herein to any soybeangene pool or population of interest, and using any particular softwareand software parameters. Indeed, observations regarding tolerancemarkers and chromosome intervals in populations in additions to thosedescribed herein are readily made using the teaching of the presentdisclosure.

Mapping Populations

Any suitable soybean strains can be used to generate mapping data or formarker association studies. A large number of commonly used soybeanlines (e.g., commercial varieties) and mapping populations are known inthe art. A broad range of mapping populations was used in the currentstudy, including, but not limited to those listed in FIG. 7.

Mapping Software

A variety of commercial software is available for genetic mapping andmarker association studies (e.g., QTL mapping). This software includesbut is not limited to:

Software Description/References JoinMap ® VanOoijen, & Voorrips (2001)“JoinMap 3.0 software for the calculation of genetic linkage maps,”Plant Research International, Wageningen, the Netherlands; and, Stam“Construction of integrated genetic linkage maps by means of a newcomputer package: JoinMap” The Plant Journal 3(5): 739-744 (1993)MapQTL ® J. W. vanOoijen, “Software for the mapping of quantitativetrait loci in experimental populations” Kyazma B. V., Wageningen,Netherlands MapManager QT Manly & Olson, “Overview of QTL mappingsoftware and introduction to Map Manager QT” Mamm. Genome 10: 327-334(1999) MapManager QTX Manly, Cudmore & Meer, “MapManager QTX,cross-platform software for genetic mapping” Mamm. Genome 12: 930-932(2001) GeneFlow ® and GENEFLOW, Inc. (Alexandria, VA) QTLocate ™ TASSEL(Trait Analysis by aSSociation, Evolution, and Linkage) by EdwardBuckler, and information about the program can be found on the BucklerLab web page at the Institute for Genomic Diversity at CornellUniversity.Unified Genetic Maps

“Unified”, “consensus” or “integrated” genetic maps have been createdthat incorporate mapping data from two or more sources, includingsources that used different mapping populations and different modes ofstatistical analysis. The merging of genetic map information increasesthe marker density on the map, as well as improving map resolution.These improved maps can be advantageously used in marker assistedselection, map-based cloning, provide an improved framework forpositioning newly identified molecular markers and aid in theidentification of QTL chromosome intervals and clusters ofadvantageously-linked markers.

In some aspects, a consensus map is derived by simply overlaying one mapon top of another. In other aspects, various algorithms, e.g., JoinMap®analysis, allows the combination of genetic mapping data from multiplesources, and reconciles discrepancies between mapping data from theoriginal sources. See, Van Ooijen & Voorrips (2001) “JoinMap 3.0software for the calculation of genetic linkage maps,” Plant ResearchInternational, Wageningen, the Netherlands; and, Stam (1993)“Construction of integrated genetic linkage maps by means of a newcomputer package: JoinMap,” The Plant Journal 3(5):739-744.

FIG. 6 provides a composite genetic map that incorporates mappinginformation from various sources. The markers that are on this map areknown in the art (i.e., have been previously described; see, e.g., theSOYBASE on-line resource for extensive listings of these markers anddescriptions of the individual markers) or are described herein.

Additional integrated maps are known in the art. See, e.g., Cregan etal., (1999) “An Integrated Genetic Linkage Map of the Soybean Genome”,Crop Sci 39:1464-1490; and also, International Application NumberPCT/US2004/024919 by Sebastian, filed Jul. 27, 2004, entitled “SoybeanPlants Having Superior Agronomic Performance and Methods for theirProduction”.

Song, et al., provides another integrated soybean genetic map thatincorporates mapping information from five different mapping populations(Song et al., (2004) “A New Integrated Genetic Linkage Map of theSoybean,” Theor Appl Genet 109:122-128). This integrated map containsapproximately 1,800 soybean markers, including SSR and SNP-type markers,as well as EST markers, RFLP markers, AFLP, RAPD, isozyme and classicalmarkers (e.g., seed coat color). The markers that are on this map areknown in the art and have been previously characterized. Thisinformation is also available at the website for the Soybean Genomicsand Improvement Laboratory (SGIL) at the USDA Beltsville AgriculturalResearch Center (BARC). See, specifically, the description of projectsin the Cregan Laboratory on that website.

The soybean integrated linkage map provided in Song et al., (2004) isbased on the principle described by Stam (1993) “Construction ofintegrated genetic linkage maps by means of a new computer package:JoinMap,” Plant J 3:739-744; and Van Ooijen & Voorrips (2001) “JoinMap3.0 software for the calculation of genetic linkage maps,” PlantResearch International, Wageningen, the Netherlands. Mapping informationfrom five soybean populations was used in the map integration, and alsoused to place recently identified SSR markers onto the soybean genome.These mapping populations were Minsoy×Noir 1 (MN), Minsoy×Archer (MA),Noir 1×Archer (NA), Clark×Harosoy (CH) and A81-356022×P1468916 (MS). TheJoinMap® analysis resulted in a map with 20 linkage groups containing atotal of 1849 markers, including 1015 SSRs, 709 RFLPs, 73 RAPDs, 24classical traits, six AFLPs, ten isozymes and 12 others. Among themapped SSR markers were 417 previously uncharacterized SSRs.

Initially, LOD scores and pairwise recombination frequencies betweenmarkers were calculated. A LOD of 5.0 was used to create groups in theMS, MA, NA populations and LOD 4.0 in the MN and CH populations. The mapof each linkage group was then integrated. Recombination values wereconverted to genetic distances using the Kosambi mapping function.

Linked Markers

From the present disclosure and widely recognized in the art, it isclear that any genetic marker that has a significant probability ofco-segregation with a phenotypic trait of interest (e.g., in the presentcase, a tolerance or improved tolerance trait) can be used as a markerfor that trait. A list of useful QTL markers provided by the presentinvention is provided in FIG. 1.

In addition to the QTL markers noted in FIG. 1, additional markerslinked to (showing linkage disequilibrium with) the QTL markers can alsobe used to predict the tolerance or improved tolerance trait in asoybean plant. In other words, any other marker showing less than 50%recombination frequency (separated by a genetic distance less than 50cM) with a QTL marker of the invention (e.g., the markers provided inFIG. 1) is also a feature of the invention. Any marker that is linked toa QTL marker can also be used advantageously in marker-assistedselection for the particular trait.

Genetic markers that are linked to QTL markers (e.g., QTL markersprovided in FIG. 1) are particularly useful when they are sufficientlyproximal (e.g., closely linked) to a given QTL marker so that thegenetic marker and the QTL marker display a low recombination frequency.In the present invention, such closely linked markers are a feature ofthe invention. As defined herein, closely linked markers display arecombination frequency of about 10% or less (the given marker is within10 cM of the QTL). Put another way, these closely linked locico-segregate at least 90% of the time. Indeed, the closer a marker is toa QTL marker, the more effective and advantageous that marker becomes asan indicator for the desired trait.

Thus, in other embodiments, closely linked loci such as a QTL markerlocus and a second locus display an inter-locus cross-over frequency ofabout 10% or less, preferably about 9% or less, still more preferablyabout 8% or less, yet more preferably about 7% or less, still morepreferably about 6% or less, yet more preferably about 5% or less, stillmore preferably about 4% or less, yet more preferably about 3% or less,and still more preferably about 2% or less. In highly preferredembodiments, the relevant loci (e.g., a marker locus and a target locussuch as a QTL) display a recombination a frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Thus, the loci are about 10 cM, 9cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or0.25 cM or less apart. Put another way, two loci that are localized tothe same chromosome, and at such a distance that recombination betweenthe two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be“proximal to” each other.

In some aspects, linked markers (including closely linked markers) ofthe invention are determined by review of a genetic map, for example,the integrated genetic map shown in FIG. 6. For example, it is shownherein that the linkage group LG-C2 marker Sct_028 correlates with atleast one Charcoal Rot Drought Complex tolerance QTL. Markers that arelinked to Sct_028 can be determined from the map provided in FIG. 6. Forexample, SSR markers on linkage group LG-C2 that are linked to Sct_028include:

Marker Map Position Satt286 101.75 Sat_402 103.33 Satt277 107.58 Satt365111.68 Satt205 112.18 Satt557 112.19 Satt289 112.34 Satt134 112.83Sat_312 112.84 Satt489 113.38 Satt319 113.41 Satt658 113.62 AG36 113.69Satt100 113.95 Sat_251 114.19 Sat_142 115.09 Satt708 115.48 Sat_238117.45 Satt460 117.76 Satt079 117.87 Sat_263 118.77 Staga001 119.84Satt307 121.26 Sct_028 122.01 Satt202 126.23 Sat_252 127.00 Satt316127.66 Satt433 128.22 Satt371 145.47

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toSct_028 on linkage group LG-C2 include:

Marker Map Position Satt205 112.18 Satt557 112.19 Satt289 112.34 Satt134112.83 Sat_312 112.84 Satt489 113.38 Satt319 113.41 Satt658 113.62 AG36113.69 Satt100 113.95 Sat_251 114.19 Sat_142 115.09 Satt708 115.48Sat_238 117.45 Satt460 117.76 Satt079 117.87 Sat_263 118.77 Staga001119.84 Satt307 121.26 Sct_028 122.01 Satt202 126.23 Sat_252 127.00Satt316 127.66 Satt433 128.22

Markers that are linked to Satt512 can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-E thatare linked to Satt512 include:

Marker Map Position Satt575 3.30 Satt213 3.72 Sat_112 8.67 Satt411 12.92Sat_124 15.86 Satt512 16.73 Satt384 19.29 Satt691 19.70 Satt720 20.79Satt651 32.09 Satt212 32.27 Satt598 34.20 Satt573 35.79 Sat_136 39.16

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toSatt512 on linkage group LG-E include:

Marker Map Position Sat_112 8.67 Satt411 12.92 Sat_124 15.86 Satt51216.73 Satt384 19.29 Satt691 19.70 Satt720 20.79

Markers that are linked to S60211-TB can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-B2 thatare linked to S60211-TB include:

Marker Map Position Satt467 17.77 Sat_342 20.30 Satt126 27.62 Sat_28731.87 S60211-TB 36.51 Sct_034 51.45 Satt083 51.49 Satt168 55.20 Satt41656.95

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toS60211-TB on linkage group LG-B2 include:

Marker Map Position Satt126 27.62 Sat_287 31.87 S60211-TB 36.51

Markers that are linked to Sat_117 can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-G thatare linked to Sat_117 include:

Marker Map Position Satt612 80.37 AF162283 87.94 Sct_199 94.40 Satt47294.83 Satt191 96.57 Sat_117 100.00 Sct_187 107.11 Sat_372 107.75 Sat_064108.69

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toSat_117 on linkage group LG-G include:

Marker Map Position Sct_199 94.40 Satt472 94.83 Satt191 96.57 Sat_117100.00 Sct_187 107.11 Sat_372 107.75 Sat_064 108.69

Markers that are linked to P13158A can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-H thatare linked to P13158A include:

Marker Map Position Sat_158 73.45 Satt302 81.04 Sat_175 83.19 Sat_21685.26 Satt637 85.79 Satt142 86.48 Satt293 89.08 Satt317 89.51 Satt18191.12 P13158A 96.00 Sat_218 99.50 Sat_180 104.37 Satt434 105.73

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toP13158A on linkage group LG-H include:

Marker Map Position Satt142 86.48 Satt293 89.08 Satt317 89.51 Satt18191.12 P13158A 96.00 Sat_218 99.50 Sat_180 104.37 Satt434 105.73

Markers that are linked to S63880-CB can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-B1 thatare linked to S63880-CB include:

Marker Map Position Satt444 85.91 Satt665 96.36 Sat_123 100.87 Satt359102.55 S63880-CB ~107 Satt484 118.52 Satt453 123.95 Sat_331 125.73

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toS63880-CB on linkage group LG-B1 include:

Marker Map Position Sat_123 100.87 Satt359 102.55 S63880-CB ~107 Satt484118.52

Markers that are linked to S00415-1-A can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-C1 thatare linked to S00799-1-A include:

Marker Map Position Bng019_1 53.86 K472_1 53.91 V38a 54.18 Satt578 65.08Satt607 67.02 A519_3 69.30 Bng140_1 69.68 Satt646 70.51 Bng161_1 70.57Dia 71.08 S00415-1-A 71.24 L192_1 73.16 Satt190 73.32 Satt161 73.38Satt718 73.79 Sat_404 73.84 Satt661 74.36 Satt139 74.45 AW277661 74.79Satt136 75.11 Satt361 75.51 Sat_077 76.00 Satt399 76.23 Sat_416 76.41Sat_357 76.43 G214_25 76.43 Sat_085 76.91 G214_24 77.26 Satt294 78.65Sat_322 79.26 Satt476 80.62 Sat_042 82.51 Satt195 84.80 Bng143_1 85.08Satt670 85.37 Sat_207 87.30 Satt713 88.94 Sat_311 90.11 A063_1 90.72Sct_191 92.98

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toS00415-1-A on linkage group LG-C1 include:

Marker Map Position Satt607 67.02 A519_3 69.30 Bng140_1 69.68 Satt64670.51 Bng161_1 70.57 Dia 71.08 S00799-1-A 71.24 L192_1 73.16 Satt19073.32 Satt161 73.38 Satt718 73.79 Sat_404 73.84 Satt661 74.36 Satt13974.45 AW277661 74.79 Satt136 75.11 Satt361 75.51 Sat_077 76.00 Satt39976.23 Sat_416 76.41 Sat_357 76.43 G214_25 76.43 Sat_085 76.91 G214_2477.26

Markers that are linked to S00705-1-A can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-D1 bthat are linked to S00705-1-A include:

Marker Map Position A605_1 64.91 Sat_423 67.62 A747_1 69.18 Sat_13570.65 Satt412 72.57 Satt141 72.88 Satt290 73.34 Satt611 74.01 Satt60474.20 K011_4 74.55 Satt506 74.79 Satt005 75.29 Satt600 75.41 L050_375.44 Satt537 75.66 Satt579 75.94 Satt282 76.09 Sat_089 76.27 Satt18976.32 Satt350 76.59 Satt428 77.34 Mng137_1 77.55 Bng047_1 77.87 Sat_16978.44 Satt644 79.41 S00705-1-A 83.80 Satt041 84.04 RGA_1f 85.14 Satt54687.19 M7E8mr2 87.80 B194_2 88.36 Sat_139 93.34 Satt703 98.75 Satt172100.88 Sat_069 102.59 Idh1 105.41 A519_2 107.61

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toS00705-1-A on linkage group LG-D1b include:

Marker Map Position Satt428 77.34 Mng137_1 77.55 Bng047_1 77.87 Sat_16978.44 Satt644 79.41 S00705-1-A 83.80 Satt041 84.04 RGA_1f 85.14 Satt54687.19 M7E8mr2 87.80 B194_2 88.36

Markers that are linked to S02118-1-A can be determined from the mapprovided in FIG. 6. For example, SSR markers on linkage group LG-N thatare linked to S02118-1-A include:

Marker Map Position Sat_306 93.11 Sat_295 95.00 Satt022 102.05 Sat_125103.33 S02118-1-A 105.63 A455_2 113.48 A363_3 116.66

In other aspects, closely linked markers of the invention can bedetermined by review of this same genetic map. For example, SSR markersthat are closely linked (e.g., separated by not more than 10 cM) toS02118-1-A on linkage group LG-N include:

Marker Map Position Satt022 102.05 Sat_125 103.33 S02118-1-A 105.63A455_2 113.48 A363_3 116.66

Similarly, linked markers (including closely linked markers) of theinvention can be determined by review of any suitable soybean geneticmap. For example, the integrated genetic map described in Song, et al.,(2004) also provides a means to identify linked (including closelylinked) markers. See, Song, et al., (2004) “A New Integrated GeneticLinkage Map of the Soybean” Theor Appl Genet 109:122-128; see also thewebsite for the Soybean Genomics and Improvement Laboratory (SGIL) atthe USDA Beltsville Agricultural Research Center (BARC), and seespecifically the description of projects in the Cregan Laboratory onthat website. That genetic map incorporates a variety of genetic markersthat are known in the art or alternatively are described in thatreference. Detailed descriptions of numerous markers, including many ofthose described in Song, et al., (2004) can be found at the SOYBASEwebsite resource.

It is not intended that the determination of linked or closely linkedmarkers be limited to the use of any particular soybean genetic map.Indeed, a large number of soybean genetic maps are available and arewell known to one of skill in the art. Another map that finds use withthe invention in this respect is the integrated soybean genetic mapsfound on the SOYBASE website resource. Alternatively still, thedetermination of linked and closely linked markers can be made by thegeneration of an experimental dataset and linkage analysis.

It is not intended that the identification of markers that are linked(e.g., within about 50 cM or within about 10 cM) to the Charcoal RotDrought Complex tolerance QTL markers identified herein be limited toany particular map or methodology. The integrated genetic map providedin FIG. 6 serves only as example for identifying linked markers. Indeed,linked markers as defined herein can be determined from any genetic mapknown in the art (an experimental map or an integrated map), oralternatively, can be determined from any new mapping dataset.

It is noted that lists of linked and closely linked markers may varybetween maps and methodologies due to various factors. First, themarkers that are placed on any two maps may not be identical, andfurthermore, some maps may have a greater marker density than anothermap. Also, the mapping populations, methodologies and algorithms used toconstruct genetic maps can differ. One of skill in the art recognizesthat one genetic map is not necessarily more or less accurate thananother, and furthermore, recognizes that any soybean genetic map can beused to determine markers that are linked and closely linked to the QTLmarkers of the present invention.

Techniques for Marker Detection

The invention provides molecular markers that have a significantprobability of co-segregation with QTL that impart a Charcoal RotDrought Complex tolerance phenotype. These QTL markers find use inmarker assisted selection for desired traits (tolerance or improvedtolerance), and also have other uses. It is not intended that theinvention be limited to any particular method for the detection of thesemarkers.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by numerous methods well-established in theart (e.g., PCR-based sequence specific amplification, restrictionfragment length polymorphisms (RFLPs), isozyme markers, allele specifichybridization (ASH), amplified variable sequences of the plant genome,self-sustained sequence replication, simple sequence repeat (SSR),single nucleotide polymorphism (SNP), random amplified polymorphic DNA(“RAPD”) or amplified fragment length polymorphisms (AFLP)). In oneadditional embodiment, the presence or absence of a molecular marker isdetermined simply through nucleotide sequencing of the polymorphicmarker region. This method is readily adapted to high throughputanalysis as are the other methods noted above, e.g., using availablehigh throughput sequencing methods such as sequencing by hybridization.

In general, the majority of genetic markers rely on one or more propertyof nucleic acids for their detection. For example, some techniques fordetecting genetic markers utilize hybridization of a probe nucleic acidto nucleic acids corresponding to the genetic marker (e.g., amplifiednucleic acids produced using genomic soybean DNA as a template).Hybridization formats, including but not limited to solution phase,solid phase, mixed phase, or in situ hybridization assays are useful forallele detection. An extensive guide to the hybridization of nucleicacids is found in Tijssen (1993) Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes Elsevier,New York; Berger & Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(“Berger”); as well as in Sambrook, and in Ausubel (herein).

For example, markers that comprise restriction fragment lengthpolymorphisms (RFLP) are detected, e.g., by hybridizing a probe which istypically a sub-fragment (or a synthetic oligonucleotide correspondingto a sub-fragment) of the nucleic acid to be detected to restrictiondigested genomic DNA. The restriction enzyme is selected to providerestriction fragments of at least two alternative (or polymorphic)lengths in different individuals or populations. Determining one or morerestriction enzyme that produces informative fragments for each cross isa simple procedure, well known in the art. After separation by length inan appropriate matrix (e.g., agarose or polyacrylamide) and transfer toa membrane (e.g., nitrocellulose, nylon, etc.), the labeled probe ishybridized under conditions which result in equilibrium binding of theprobe to the target followed by removal of excess probe by washing.

Nucleic acid probes to the marker loci can be cloned and/or synthesized.Any suitable label can be used with a probe of the invention. Detectablelabels suitable for use with nucleic acid probes include, for example,any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels include biotin for staining with labeledstreptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels,enzymes, and colorimetric labels. Other labels include ligands whichbind to antibodies labeled with fluorophores, chemiluminescent agents,and enzymes. A probe can also constitute radiolabelled PCR primers thatare used to generate a radiolabelled amplicon. Labeling strategies forlabeling nucleic acids and corresponding detection strategies can befound, e.g., in Haugland (1996) Handbook of Fluorescent Probes andResearch Chemicals Sixth Edition by Molecular Probes, Inc. (EugeneOreg.); or Haugland (2001) Handbook of Fluorescent Probes and ResearchChemicals Eighth Edition by Molecular Probes, Inc. (Eugene Oreg.)(Available on CD ROM).

Amplification-Based Detection Methods

PCR, RT-PCR and LCR are in particularly broad use as amplification andamplification-detection methods for amplifying nucleic acids of interest(e.g., those comprising marker loci), facilitating detection of themarkers. Details regarding the use of these and other amplificationmethods can be found in any of a variety of standard texts, including,e.g., Sambrook, Ausubel, Berger and Croy, herein. Many available biologytexts also have extended discussions regarding PCR and relatedamplification methods. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase (“Reverse Transcription-PCR, or “RT-PCR”). See alsoAusubel, Sambrook and Berger, above.

Real Time Amplification/Detection Methods

In one aspect, real time PCR or LCR is performed on the amplificationmixtures described herein, e.g., using molecular beacons or TaqMan™probes. A molecular beacon (MB) is an oligonucleotide or PNA which,under appropriate hybridization conditions, self-hybridizes to form astem and loop structure. The MB has a label and a quencher at thetermini of the oligonucleotide or PNA; thus, under conditions thatpermit intra-molecular hybridization, the label is typically quenched(or at least altered in its fluorescence) by the quencher. Underconditions where the MB does not display intra-molecular hybridization(e.g., when bound to a target nucleic acid, e.g., to a region of anamplicon during amplification), the MB label is unquenched. Detailsregarding standard methods of making and using MBs are well establishedin the literature and MBs are available from a number of commercialreagent sources. See also, e.g., Leone, et al., (1995) “Molecular beaconprobes combined with amplification by NASBA enable homogenous real-timedetection of RNA” Nucl Acids Res 26:2150-2155; Tyagi & Kramer, (1996)“Molecular beacons: probes that fluoresce upon hybridization” NatureBiotechnology 14:303-308; Blok & Kramer, (1997) “Amplifiablehybridization probes containing a molecular switch” Mol Cell Probes11:187-194; Hsuih et al. (1997) “Novel, ligation-dependent PCR assay fordetection of hepatitis C in serum” J Clin Microbiol 34:501-507;Kostrikis et al. (1998) “Molecular beacons: spectral genotyping of humanalleles” Science 279:1228-1229; Sokol et al. (1998) “Real time detectionof DNA:RNA hybridization in living cells” Proc Natl Acad Sci USA95:11538-11543; Tyagi et al. (1998) “Multicolor molecular beacons forallele discrimination” Nat Biotechnol 16:49-53; Bonnet et al. (1999)“Thermodynamic basis of the chemical specificity of structured DNAprobes” Proc Natl Acad Sci USA 96:6171-6176; Fang et al. (1999)“Designing a novel molecular beacon for surface-immobilized DNAhybridization studies” J Am Chem Soc 121:2921-2922; Marras et al. (1999)“Multiplex detection of single-nucleotide variation using molecularbeacons” Genet Anal Biomol Eng 14:151-156; and Vet et al. (1999)“Multiplex detection of four pathogenic retroviruses using molecularbeacons” Proc Natl Acad Sci USA 96:6394-6399. Additional detailsregarding MB construction and use is found in the patent literature,e.g., U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al., entitled“Detectably labeled dual conformation oligonucleotide probes, assays andkits;” U.S. Pat. No. 6,150,097 (Nov. 21, 2000) to Tyagi et al., entitled“Nucleic acid detection probes having non-FRET fluorescence quenchingand kits and assays including such probes” and U.S. Pat. No. 6,037,130(Mar. 14, 2000) to Tyagi et al., entitled “Wavelength-shifting probesand primers and their use in assays and kits.”

PCR detection and quantification using dual-labeled fluorogenicoligonucleotide probes, commonly referred to as “TagMan™” probes, canalso be performed according to the present invention. These probes arecomposed of short (e.g., 20-25 base) oligodeoxynucleotides that arelabeled with two different fluorescent dyes. On the 5′ terminus of eachprobe is a reporter dye, and on the 3′ terminus of each probe aquenching dye is found. The oligonucleotide probe sequence iscomplementary to an internal target sequence present in a PCR amplicon.When the probe is intact, energy transfer occurs between the twofluorophores and emission from the reporter is quenched by the quencherby FRET. During the extension phase of PCR, the probe is cleaved by 5′nuclease activity of the polymerase used in the reaction, therebyreleasing the reporter from the oligonucleotide-quencher and producingan increase in reporter emission intensity. Accordingly, TaqMan™ probesare oligonucleotides that have a label and a quencher, where the labelis released during amplification by the exonuclease action of thepolymerase used in amplification. This provides a real time measure ofamplification during synthesis. A variety of TaqMan™ reagents arecommercially available, e.g., from Applied Biosystems (DivisionHeadquarters in Foster City, Calif.) as well as from a variety ofspecialty vendors such as Biosearch Technologies (e.g., black holequencher probes).

Additional Details Regarding Amplified Variable Sequences, SSR, AFLPASH, SNPs and Isozyme Markers

Amplified variable sequences refer to amplified sequences of the plantgenome which exhibit high nucleic acid residue variability betweenmembers of the same species. All organisms have variable genomicsequences and each organism (with the exception of a clone) has adifferent set of variable sequences. Once identified, the presence ofspecific variable sequence can be used to predict phenotypic traits.Preferably, DNA from the plant serves as a template for amplificationwith primers that flank a variable sequence of DNA. The variablesequence is amplified and then sequenced.

Alternatively, self-sustained sequence replication can be used toidentify genetic markers. Self-sustained sequence replication refers toa method of nucleic acid amplification using target nucleic acidsequences which are replicated exponentially in vitro undersubstantially isothermal conditions by using three enzymatic activitiesinvolved in retroviral replication: (1) reverse transcriptase, (2) RNaseH, and (3) a DNA-dependent RNA polymerase (Guatelli et al. (1990) ProcNatl Acad Sci USA 87:1874). By mimicking the retroviral strategy of RNAreplication by means of cDNA intermediates, this reaction accumulatescDNA and RNA copies of the original target.

Amplified fragment length polymorphisms (AFLP) can also be used asgenetic markers (Vos, et al., (1995) Nucleic Acids Res 23:4407). Thephrase “amplified fragment length polymorphism” refers to selectedrestriction fragments which are amplified before or after cleavage by arestriction endonuclease. The amplification step allows easier detectionof specific restriction fragments. AFLP allows the detection largenumbers of polymorphic markers and has been used for genetic mapping ofplants (Becker et al. (1995) Mol Gen Genet 249:65; and Meksem et al.(1995) Mol Gen Genet 249:74).

Allele-specific hybridization (ASH) can be used to identify the geneticmarkers of the invention. ASH technology is based on the stableannealing of a short, single-stranded, oligonucleotide probe to acompletely complementary single-strand target nucleic acid. Detection isvia an isotopic or non-isotopic label attached to the probe.

For each polymorphism, two or more different ASH probes are designed tohave identical DNA sequences except at the polymorphic nucleotides. Eachprobe will have exact homology with one allele sequence so that therange of probes can distinguish all the known alternative allelesequences. Each probe is hybridized to the target DNA. With appropriateprobe design and hybridization conditions, a single-base mismatchbetween the probe and target DNA will prevent hybridization. In thismanner, only one of the alternative probes will hybridize to a targetsample that is homozygous or homogenous for an allele. Samples that areheterozygous or heterogeneous for two alleles will hybridize to both oftwo alternative probes.

ASH markers are used as dominant markers where the presence or absenceof only one allele is determined from hybridization or lack ofhybridization by only one probe. The alternative allele may be inferredfrom the lack of hybridization. ASH probe and target molecules areoptionally RNA or DNA; the target molecules are any length ofnucleotides beyond the sequence that is complementary to the probe; theprobe is designed to hybridize with either strand of a DNA target; theprobe ranges in size to conform to variously stringent hybridizationconditions, etc.

PCR allows the target sequence for ASH to be amplified from lowconcentrations of nucleic acid in relatively small volumes. Otherwise,the target sequence from genomic DNA is digested with a restrictionendonuclease and size separated by gel electrophoresis. Hybridizationstypically occur with the target sequence bound to the surface of amembrane or, as described in U.S. Pat. No. 5,468,613, the ASH probesequence may be bound to a membrane.

In one embodiment, ASH data are typically obtained by amplifying nucleicacid fragments (amplicons) from genomic DNA using PCR, transferring theamplicon target DNA to a membrane in a dot-blot format, hybridizing alabeled oligonucleotide probe to the amplicon target, and observing thehybridization dots by autoradiography.

Single nucleotide polymorphisms (SNP) are markers that consist of ashared sequence differentiated on the basis of a single nucleotide.Typically, this distinction is detected by differential migrationpatterns of an amplicon comprising the SNP on e.g., an acrylamide gel.However, alternative modes of detection, such as hybridization, e.g.,ASH, or RFLP analysis are also appropriate.

Isozyme markers can be employed as genetic markers, e.g., to trackmarkers other than the tolerance markers herein, or to track isozymemarkers linked to the markers herein. Isozymes are multiple forms ofenzymes that differ from one another in their amino acid, and thereforetheir nucleic acid sequences. Some isozymes are multimeric enzymescontaining slightly different subunits. Other isozymes are eithermultimeric or monomeric but have been cleaved from the proenzyme atdifferent sites in the amino acid sequence. Isozymes can becharacterized and analyzed at the protein level, or alternatively,isozymes which differ at the nucleic acid level can be determined. Insuch cases any of the nucleic acid based methods described herein can beused to analyze isozyme markers.

Additional Details Regarding Nucleic Acid Amplification

As noted, nucleic acid amplification techniques such as PCR and LCR arewell known in the art and can be applied to the present invention toamplify and/or detect nucleic acids of interest, such as nucleic acidscomprising marker loci. Examples of techniques sufficient to directpersons of skill through such in vitro methods, including the polymerasechain reaction (PCR), the ligase chain reaction (LCR), Q1313-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), are found in the references noted above, e.g., Innis, Sambrook,Ausubel, Berger and Croy. Additional details are found in Mullis, etal., (1987) U.S. Pat. No. 4,683,202; Arnheim & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3:81-94; Kwoh et al.(1989) Proc Natl Acad Sci USA 86:1173; Guatelli et al. (1990) Proc NatlAcad Sci USA 87:1874; Lomeli et al. (1989) J Clin Chem 35:1826;Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990)Biotechnology 8:291-294; Wu & Wallace (1989) Gene 4:560; Barringer etal. (1990) Gene 89:117, and Sooknanan & Malek, (1995) Biotechnology13:563-564. Improved methods of amplifying large nucleic acids by PCR,which is useful in the context of positional cloning, are furthersummarized in Cheng, et al., (1994) Nature 369:684, and the referencestherein, in which PCR amplicons of up to 40 kb are generated.

Probe/Primer Synthesis Methods

In general, synthetic methods for making oligonucleotides, includingprobes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids),etc., are well known. For example, oligonucleotides can be synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, (1981) Tetrahedron Letts22(20):1859-1862, e.g., using a commercially available automatedsynthesizer, e.g., as described in Needham-VanDevanter et al. (1984)Nucl Acids Res 12:6159-6168. Oligonucleotides, including modifiedoligonucleotides can also be ordered from a variety of commercialsources known to persons of skill. There are many commercial providersof oligo synthesis services, and thus this is a broadly accessibletechnology. Any nucleic acid can be custom ordered from any of a varietyof commercial sources, such as The Midland Certified Reagent Company,The Great American Gene Company, ExpressGen Inc., Operon TechnologiesInc. (Alameda, Calif.) and many others. Similarly, PNAs can be customordered from any of a variety of sources, such as PeptidoGenic, HTIBio-Products, Inc., BMA Biomedicals Ltd (U.K.), Bio•Synthesis, Inc., andmany others.

In Silico Marker Detection

In alternative embodiments, in silico methods can be used to detect themarker loci of interest. For example, the sequence of a nucleic acidcomprising the marker locus of interest can be stored in a computer. Thedesired marker locus sequence or its homolog can be identified using anappropriate nucleic acid search algorithm as provided by, for example,in such readily available programs as BLAST, or even simple wordprocessors.

Amplification Primers for Marker Detection

In some preferred embodiments, the molecular markers of the inventionare detected using a suitable PCR-based detection method, where the sizeor sequence of the PCR amplicon is indicative of the absence or presenceof the marker (e.g., a particular marker allele). In these types ofmethods, PCR primers are hybridized to the conserved regions flankingthe polymorphic marker region. As used in the art, PCR primers used toamplify a molecular marker are sometimes termed “PCR markers” or simply“markers”.

It will be appreciated that, although many specific examples of primersare provided herein (see, FIG. 2), suitable primers to be used with theinvention can be designed using any suitable method. It is not intendedthat the invention be limited to any particular primer or primer pair.For example, primers can be designed using any suitable softwareprogram, such as LASERGENE®.

In some embodiments, the primers of the invention are radiolabelled, orlabeled by any suitable means (e.g., using a non-radioactive fluorescenttag), to allow for rapid visualization of the different size ampliconsfollowing an amplification reaction without any additional labeling stepor visualization step. In some embodiments, the primers are not labeled,and the amplicons are visualized following their size resolution, e.g.,following agarose gel electrophoresis. In some embodiments, ethidiumbromide staining of the PCR amplicons following size resolution allowsvisualization of the different size amplicons.

It is not intended that the primers of the invention be limited togenerating an amplicon of any particular size. For example, the primersused to amplify the marker loci and alleles herein are not limited toamplifying the entire region of the relevant locus. The primers cangenerate an amplicon of any suitable length that is longer or shorterthan those given in the allele definitions in FIG. 4. In someembodiments, marker amplification produces an amplicon at least 20nucleotides in length, or alternatively, at least 50 nucleotides inlength, or alternatively, at least 100 nucleotides in length, oralternatively, at least 200 nucleotides in length. Marker alleles inaddition to those recited in FIG. 4 also find use with the presentinvention.

Marker Assisted Selection and Breeding of Plants

A primary motivation for development of molecular markers in cropspecies is the potential for increased efficiency in plant breedingthrough marker assisted selection (MAS). Genetic markers are used toidentify plants that contain a desired genotype at one or more loci, andthat are expected to transfer the desired genotype, along with a desiredphenotype to their progeny. Genetic markers can be used to identifyplants that contain a desired genotype at one locus, or at severalunlinked or linked loci (e.g., a haplotype), and that would be expectedto transfer the desired genotype, along with a desired phenotype totheir progeny. The present invention provides the means to identifyplants, particularly soybean plants, that are tolerant, exhibit improvedtolerance or are susceptible to Charcoal Rot Drought Complex byidentifying plants having a specified allele at one of those loci, e.g.,Sct_028, Satt512, S60211-TB, Sat_117, P13158A, 563880-CB, S00415-1-A,S00705-1-A, and S02118-1-A.

Similarly, by identifying plants lacking the desired marker locus,susceptible or less tolerant plants can be identified, and, e.g.,eliminated from subsequent crosses. Similarly, these marker loci can beintrogressed into any desired genomic background, germplasm, plant,line, variety, etc., as part of an overall MAS breeding program designedto enhance soybean yield.

The invention also provides chromosome QTL intervals that find equal usein MAS to select plants that demonstrate Charcoal Rot Drought Complextolerance or improved tolerance. Similarly, the QTL intervals can alsobe used to counter-select plants that are susceptible or have reducedtolerance to Charcoal Rot Drought Complex. Any marker that maps withinthe QTL interval (including the termini of the intervals) finds use withthe invention. These intervals are defined by the following pairs ofmarkers:

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1)    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N).

In general, MAS uses polymorphic markers that have been identified ashaving a significant likelihood of co-segregation with a tolerancetrait. Such markers are presumed to map near a gene or genes that givethe plant its tolerance phenotype, and are considered indicators for thedesired trait, and are termed QTL markers. Plants are tested for thepresence of a desired allele in the QTL marker. The most preferredmarkers (or marker alleles) are those that have the strongestassociation with the tolerance trait.

Linkage analysis is used to determine which polymorphic marker alleledemonstrates a statistical likelihood of co-segregation with thetolerance phenotype (thus, a “tolerance marker allele”). Followingidentification of a marker allele for co-segregation with the tolerancephenotype, it is possible to use this marker for rapid, accuratescreening of plant lines for the tolerance allele without the need togrow the plants through their life cycle and await phenotypicevaluations, and furthermore, permits genetic selection for theparticular tolerance allele even when the molecular identity of theactual tolerance QTL is unknown. Tissue samples can be taken, forexample, from the first leaf of the plant and screened with theappropriate molecular marker, and it is rapidly determined which progenywill advance. Linked markers also remove the impact of environmentalfactors that can often influence phenotypic expression.

A polymorphic QTL marker locus can be used to select plants that containthe marker allele (or alleles) that correlate with the desired tolerancephenotype. In brief, a nucleic acid corresponding to the marker nucleicacid allele is detected in a biological sample from a plant to beselected. This detection can take the form of hybridization of a probenucleic acid to a marker allele or amplicon thereof, e.g., usingallele-specific hybridization, Southern analysis, northern analysis, insitu hybridization, hybridization of primers followed by PCRamplification of a region of the marker, or the like. A variety ofprocedures for detecting markers are described herein, e.g., in thesection entitled “TECHNIQUES FOR MARKER DETECTION.” After the presence(or absence) of a particular marker allele in the biological sample isverified, the plant is selected, e.g., used to make progeny plants byselective breeding.

Soybean plant breeders desire combinations of tolerance loci with genesfor high yield and other desirable traits to develop improved soybeanvarieties. Screening large numbers of samples by non-molecular methods(e.g., trait evaluation in soybean plants) can be expensive, timeconsuming, and unreliable. Use of the polymorphic markers describedherein, when genetically-linked to tolerance loci, provide an effectivemethod for selecting resistant varieties in breeding programs. Forexample, one advantage of marker-assisted selection over fieldevaluations for tolerance resistance is that MAS can be done at any timeof year, regardless of the growing season. Moreover, environmentaleffects are largely irrelevant to marker-assisted selection.

When a population is segregating for multiple loci affecting one ormultiple traits, e.g., multiple loci involved in tolerance, or multipleloci each involved in tolerance or resistance to different diseases, theefficiency of MAS compared to phenotypic screening becomes even greater,because all of the loci can be evaluated in the lab together from asingle sample of DNA. In the present instance, the Sct_028, Satt512,S60211-TB, Sat_117, P13158A, 563880-CB, S00415-1-A, S00705-1-A, andS02118-1-A markers, as well as any of the chromosome intervals

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1);    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N).        can be assayed simultaneously or sequentially from a single        sample or a population of samples.

Another use of MAS in plant breeding is to assist the recovery of therecurrent parent genotype by backcross breeding. Backcross breeding isthe process of crossing a progeny back to one of its parents or parentlines. Backcrossing is usually done for the purpose of introgressing oneor a few loci from a donor parent (e.g., a parent comprising desirabletolerance marker loci) into an otherwise desirable genetic backgroundfrom the recurrent parent (e.g., an otherwise high yielding soybeanline). The more cycles of backcrossing that are done, the greater thegenetic contribution of the recurrent parent to the resultingintrogressed variety. This is often necessary, because tolerant plantsmay be otherwise undesirable, e.g., due to low yield, low fecundity, orthe like. In contrast, strains which are the result of intensivebreeding programs may have excellent yield, fecundity or the like,merely being deficient in one desired trait such as tolerance toCharcoal Rot Drought Complex.

The presence and/or absence of a particular genetic marker or allele,e.g., Sct_028, Satt512, S60211-TB, Sat_117, P13158A, S63880-C B,S00415-1-A, S00705-1-A, and S02118-1-A markers, as well as any of thechromosome intervals

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1);    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N)        in the genome of a plant is made by any method noted herein. If        the nucleic acids from the plant are positive for a desired        genetic marker allele, the plant can be self fertilized to        create a true breeding line with the same genotype, or it can be        crossed with a plant with the same marker or with other desired        characteristics to create a sexually crossed hybrid generation.        Introgression of Favorable Alleles—Efficient Backcrossing of        Tolerance Markers into Elite Lines

One application of MAS, in the context of the present invention is touse the tolerance or improved tolerance markers to increase theefficiency of an introgression or backcrossing effort aimed atintroducing a tolerance QTL into a desired (typically high yielding)background. In marker assisted backcrossing of specific markers (andassociated QTL) from a donor source, e.g., to an elite or exotic geneticbackground, one selects among backcross progeny for the donor trait andthen uses repeated backcrossing to the elite or exotic line toreconstitute as much of the elite/exotic background's genome aspossible.

Thus, the markers and methods of the present invention can be utilizedto guide marker assisted selection or breeding of soybean varieties withthe desired complement (set) of allelic forms of chromosome segmentsassociated with superior agronomic performance (tolerance, along withany other available markers for yield, disease resistance, etc.). Any ofthe disclosed marker alleles can be introduced into a soybean line viaintrogression, by traditional breeding (or introduced viatransformation, or both) to yield a soybean plant with superioragronomic performance. The number of alleles associated with tolerancethat can be introduced or be present in a soybean plant of the presentinvention ranges from 1 to the number of alleles disclosed herein, eachinteger of which is incorporated herein as if explicitly recited.

The present invention also extends to a method of making a progenysoybean plant and these progeny soybean plants, per se. The methodcomprises crossing a first parent soybean plant with a second soybeanplant and growing the female soybean plant under plant growth conditionsto yield soybean plant progeny. Methods of crossing and growing soybeanplants are well within the ability of those of ordinary skill in theart. Such soybean plant progeny can be assayed for alleles associatedwith tolerance and, thereby, the desired progeny selected. Such progenyplants or seed can be sold commercially for soybean production, used forfood, processed to obtain a desired constituent of the soybean, orfurther utilized in subsequent rounds of breeding. At least one of thefirst or second soybean plants is a soybean plant of the presentinvention in that it comprises at least one of the allelic forms of themarkers of the present invention, such that the progeny are capable ofinheriting the allele.

Often, a method of the present invention is applied to at least onerelated soybean plant such as from progenitor or descendant lines in thesubject soybean plant's pedigree such that inheritance of the desiredtolerance allele can be traced. The number of generations separating thesoybean plants being subject to the methods of the present inventionwill generally be from 1 to 20, commonly 1 to 5, and typically 1, 2 or 3generations of separation, and quite often a direct descendant or parentof the soybean plant will be subject to the method (i.e., one generationof separation).

Introgression of Favorable Alleles—Incorporation of “Exotic” Germplasmwhile Maintaining Breeding Progress

Genetic diversity is important for long term genetic gain in anybreeding program. With limited diversity, genetic gain will eventuallyplateau when all of the favorable alleles have been fixed within theelite population. One objective is to incorporate diversity into anelite pool without losing the genetic gain that has already been madeand with the minimum possible investment. MAS provide an indication ofwhich genomic regions and which favorable alleles from the originalancestors have been selected for and conserved over time, facilitatingefforts to incorporate favorable variation from exotic germplasm sources(parents that are unrelated to the elite gene pool) in the hopes offinding favorable alleles that do not currently exist in the elite genepool.

For example, the markers of the present invention can be used for MAS incrosses involving elite×exotic soybean lines by subjecting thesegregating progeny to MAS to maintain major yield alleles, along withthe tolerance marker alleles herein.

Generation of Transgenic Cells and Plants

The present invention also relates to host cells and organisms which aretransformed with nucleic acids corresponding to tolerance QTL identifiedaccording to the invention. For example, such nucleic acids includechromosome intervals (e.g., genomic fragments) that encode a toleranceor improved tolerance trait.

General texts which describe molecular biological techniques for thecloning and manipulation of nucleic acids and production of encodedpolypeptides include Berger, Sambrook, and Ausubel, herein. These textsdescribe mutagenesis, the use of vectors, promoters and many otherrelevant topics related to, e.g., the generation of clones that comprisenucleic acids of interest, e.g., marker loci, marker probes, QTL thatsegregate with marker loci, etc.

Host cells are genetically engineered (e.g., transduced, transfected,transformed, etc.) with the vectors of this invention which can be, forexample, a cloning vector, a shuttle vector or an expression vector.Such vectors are, for example, in the form of a plasmid, a phagemid, anAgrobacterium, a virus, a naked polynucleotide (linear or circular), ora conjugated polynucleotide. Vectors can be introduced into bacteria,especially for the purpose of propagation and expansion. The vectors arealso introduced into plant tissues, cultured plant cells or plantprotoplasts by a variety of standard methods known in the art, includingbut not limited to electroporation (Fromm et al. (1985) Proc Natl AcadSci USA 82:5824), infection by viral vectors such as cauliflower mosaicvirus (CaMV) (Hohn et al. (1982) Molecular Biology of Plant TumorsAcademic Press, New York, pp. 549-560; Howell, U.S. Pat. No. 4,407,956),high velocity ballistic penetration by small particles with the nucleicacid either within the matrix of small beads or particles, or on thesurface (Klein et al. (1987) Nature 327:70), use of pollen as vector(WO85/01856), or use of Agrobacterium tumefaciens or A. rhizogenescarrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNAplasmid is transmitted to plant cells upon infection by Agrobacteriumtumefaciens, and a portion is stably integrated into the plant genome(Horsch et al. (1984) Science 233:496; Fraley et al. (1983) Proc NatlAcad Sci USA 80:4803). Additional details regarding nucleic acidintroduction methods are found in Sambrook, Berger and Ausubel, supra.The method of introducing a nucleic acid of the present invention into ahost cell is not critical to the instant invention, and it is notintended that the invention be limited to any particular method forintroducing exogenous genetic material into a host cell. Thus, anysuitable method, e.g., including but not limited to the methods providedherein, which provides for effective introduction of a nucleic acid intoa cell or protoplast can be employed and finds use with the invention.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, activatingpromoters or selecting transformants. These cells can optionally becultured into transgenic plants. In addition to Sambrook, Berger andAusubel, supra, plant regeneration from cultured protoplasts isdescribed in Evans et al. (1983) “Protoplast Isolation and Culture,”Handbook of Plant Cell Cultures 1:124-176 (MacMillan Publishing Co., NewYork; Davey, (1983) “Recent Developments in the Culture and Regenerationof Plant Protoplasts,” Protoplasts, pp. 12-29, (Birkhauser, Basel);Dale, (1983) “Protoplast Culture and Plant Regeneration of Cereals andOther Recalcitrant Crops,” Protoplasts pp. 31-41, (Birkhauser, Basel);Binding (1985) “Regeneration of Plants,” Plant Protoplasts, pp. 21-73,(CRC Press, Boca Raton, Fla.). Additional details regarding plant cellculture and regeneration include Payne, et al., (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips, (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Plant Molecular Biology (1993) Croy, Ed. BiosScientific Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culturemedia in general are also set forth in Atlas and Parks, (eds) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Additional information for cell culture is found in available commercialliterature such as the Life Science Research Cell Culture Catalogue(1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and,e.g., the Plant Culture Catalogue and supplement (e.g., 1997 or later)also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”).

The present invention also relates to the production of transgenicorganisms, which may be bacteria, yeast, fungi, animals or plants,transduced with the nucleic acids of the invention (e.g., nucleic acidscomprising the marker loci and/or QTL noted herein). A thoroughdiscussion of techniques relevant to bacteria, unicellular eukaryotesand cell culture is found in references enumerated herein and arebriefly outlined as follows. Several well-known methods of introducingtarget nucleic acids into bacterial cells are available, any of whichmay be used in the present invention. These include: fusion of therecipient cells with bacterial protoplasts containing the DNA, treatmentof the cells with liposomes containing the DNA, electroporation,projectile bombardment (biolistics), carbon fiber delivery, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria. For their proper use, follow the manufacturer's instructions(see, for example, EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect plant cells or incorporated intoAgrobacterium tumefaciens related vectors to infect plants. Typicalvectors contain transcription and translation terminators, transcriptionand translation initiation sequences, and promoters useful forregulation of the expression of the particular target nucleic acid. Thevectors optionally comprise generic expression cassettes containing atleast one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith,(1979) Gene 8:81; Roberts et al. (1987) Nature 328:731; Schneider et al.(1995) Protein Expr Purif 6435:10; Ausubel, Sambrook, Berger (allsupra). A catalogue of Bacteria and Bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al., (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson, et al., (1992) Recombinant DNA, Second Edition,Scientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex.), The Great American Gene Company (Ramona, Calif.),ExpressGen Inc. (Chicago, Ill.), Operon Technologies Inc. (Alameda,Calif.) and many others.

Introducing Nucleic Acids into Plants.

Techniques for transforming plant cells with nucleic acids are widelyavailable and can be readily adapted to the invention. In addition toBerger, Ausubel and Sambrook, all supra, useful general references forplant cell cloning, culture and regeneration include Jones, (ed) (1995)Plant Gene Transfer and Expression Protocols—Methods in MolecularBiology, Volume 49 Humana Press Towata N.J.; Payne, et al., (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y. (Payne); and Gamborg and Phillips, (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) (Gamborg). A variety ofcell culture media are described in Atlas and Parks, (eds) The Handbookof Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas).Additional information for plant cell culture is found in availablecommercial literature such as the Life Science Research Cell CultureCatalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (Sigma-LSRCCC)and, e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St Louis, Mo.) (Sigma-PCCS). Additional detailsregarding plant cell culture are found in Croy, (ed.) (1993) PlantMolecular Biology, Bios Scientific Publishers, Oxford, U.K.

The nucleic acid constructs of the invention, e.g., plasmids, cosmids,artificial chromosomes, DNA and RNA polynucleotides, are introduced intoplant cells, either in culture or in the organs of a plant by a varietyof conventional techniques. Techniques for transforming a wide varietyof higher plant species are also well known and described in widelyavailable technical, scientific, and patent literature. See, forexample, Weissinger et al. (1988) Ann Rev Genet 22:421-477. The DNAconstructs of the invention, for example plasmids, phagemids, cosmids,phage, naked or variously conjugated-DNA polynucleotides, (e.g.,polylysine-conjugated DNA, peptide-conjugated DNA, liposome-conjugatedDNA, etc.), or artificial chromosomes, can be introduced directly intothe genomic DNA of the plant cell using techniques such aselectroporation and microinjection of plant cell protoplasts, or the DNAconstructs can be introduced directly to plant cells using ballisticmethods, such as DNA particle bombardment.

Microinjection techniques for injecting plant, e.g., cells, embryos,callus and protoplasts, are known in the art and well described in thescientific and patent literature. For example, a number of methods aredescribed in Jones, (ed) (1995) Plant Gene Transfer and ExpressionProtocols—Methods in Molecular Biology, Volume 49 Humana Press, Towata,N.J., as well as in the other references noted herein and available inthe literature.

For example, the introduction of DNA constructs using polyethyleneglycol precipitation is described in Paszkowski et al. (1984) EMBO J3:2717. Electroporation techniques are described in Fromm et al. (1985)Proc Natl Acad Sci USA 82:5824. Ballistic transformation techniques aredescribed in Klein et al. (1987) Nature 327:70-73. Additional detailsare found in Jones (1995) and Gamborg & Phillips (1995), supra, and inU.S. Pat. No. 5,990,387.

Alternatively, and in some cases preferably, Agrobacterium mediatedtransformation is employed to generate transgenic plants.Agrobacterium-mediated transformation techniques, including disarmingand use of binary vectors, are also well described in the scientificliterature. See, for example, Horsch et al. (1984) Science 233:496; andFraley et al. (1984) Proc Natl Acad Sci USA 80:4803 and recentlyreviewed in Hansen & Chilton, (1998) Current Topics in Microbiology240:22; and Das (1998) Subcellular Biochemistry 29: Plant MicrobeInteractions, pp 343-363.

DNA constructs are optionally combined with suitable T-DNA flankingregions and introduced into a conventional Agrobacterium tumefacienshost vector. The virulence functions of the Agrobacterium tumefacienshost will direct the insertion of the construct and adjacent marker intothe plant cell DNA when the cell is infected by the bacteria. See, U.S.Pat. No. 5,591,616. Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein & Fuller, (1987) In: Genetic Engineering, vol. 6, P W JRigby, Ed., London, Academic Press; and Lichtenstein & Draper (1985) In:DNA Cloning, Vol. II, Glover, Ed., Oxford, IRI Press; WO 88/02405,published Apr. 7, 1988, describes the use of A. rhizogenes strain A4 andits Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16 (2)liposome-mediated DNA uptake (see, e.g., Freeman et al. (1984) PlantCell Physiol 25:1353), (3) the vortexing method (see, e.g., Kindle(1990) Proc Natl Acad Sci USA 87:1228.

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al. (1983) Methods Enzymol 101:433; Hess(1987) Intern Rev Cytol 107:367; Luo et al. (1988) Plant Mol Biol Rep6:165. Expression of polypeptide coding genes can be obtained byinjection of the DNA into reproductive organs of a plant as described byPena et al. (1987) Nature 325:274. DNA can also be injected directlyinto the cells of immature embryos and the desiccated embryos rehydratedas described by Neuhaus et al. (1987) Theor Appl Genet 75:30; andBenbrook, et al., (1986) in Proceedings Bio Expo Butterworth, Stoneham,Mass., pp. 27-54. A variety of plant viruses that can be employed asvectors are known in the art and include cauliflower mosaic virus(CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

Generation/Regeneration of Transgenic Plants

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg & Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York); Evans et al. (1983) Protoplasts Isolation andCulture, Handbook of Plant Cell Culture pp. 124-176, MacmillianPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp. 21-73, CRC Press, Boca Raton. Regeneration canalso be obtained from plant callus, explants, somatic embryos (Dandekaret al. (1989) J Tissue Cult Meth 12:145; McGranahan et al. (1990) PlantCell Rep 8:512) organs, or parts thereof. Such regeneration techniquesare described generally in Klee et al. (1987) Ann Rev Plant Phys38:467-486. Additional details are found in Payne (1992) and Jones(1995), both supra, and Weissbach & Weissbach, eds. (1988) Methods forPlant Molecular Biology Academic Press, Inc., San Diego, Calif. Thisregeneration and growth process includes the steps of selection oftransformant cells and shoots, rooting the transformant shoots andgrowth of the plantlets in soil. These methods are adapted to theinvention to produce transgenic plants bearing QTLs according to themethods of the invention.

In addition, the regeneration of plants containing nucleic acids of thepresent invention and introduced by Agrobacterium into cells of leafexplants can be achieved as described by Horsch et al. (1985) Science227:1229-1231. In this procedure, transformants are grown in thepresence of a selection agent and in a medium that induces theregeneration of shoots in the plant species being transformed asdescribed by Fraley et al. (1983) Proc Natl Acad Sci USA 80:4803. Thisprocedure typically produces shoots within two to four weeks and thesetransformant shoots are then transferred to an appropriate root-inducingmedium containing the selective agent and an antibiotic to preventbacterial growth. Transgenic plants of the present invention may befertile or sterile.

It is not intended that plant transformation and expression ofpolypeptides that provide disease tolerance, as provided by the presentinvention, be limited to soybean species. Indeed, it is contemplatedthat the polypeptides that provide the desired tolerance in soybean canalso provide such tolerance when transformed and expressed in otheragronomically and horticulturally important species. Such speciesinclude primarily dicots, e.g., of the families: Leguminosae (includingpea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean,clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria andsweetpea); and Compositae (the largest family of vascular plants,including at least 1,000 genera, including important commercial cropssuch as sunflower).

Additionally, preferred targets for modification with the nucleic acidsof the invention, as well as those specified above, plants from thegenera: Allium, Apium, Arachis, Brassica, Capsicum, Cicer, Cucumis,Curcubita, Daucus, Fagopyrum, Glycine, Helianthus, Lactuca, Lens,Lycopersicon, Medicago, Pisum, Phaseolus, Solanum, Trifolium, Vigna andmany others.

Common crop plants which are targets of the present invention includesoybean, sunflower, canola, peas, beans, lentils, peanuts, yam beans,cowpeas, velvet beans, clover, alfalfa, lupine, vetch, sweet clover,sweetpea, field pea, fava bean, broccoli, brussel sprouts, cabbage,cauliflower, kale, kohlrabi, celery, lettuce, carrot, onion, pepper,potato, eggplant and tomato.

In construction of recombinant expression cassettes of the invention,which include, for example, helper plasmids comprising virulencefunctions, and plasmids or viruses comprising exogenous DNA sequencessuch as structural genes, a plant promoter fragment is optionallyemployed which directs expression of a nucleic acid in any or alltissues of a regenerated plant. Examples of constitutive promotersinclude the cauliflower mosaic virus (CaMV) 35S transcription initiationregion, the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumefaciens, and other transcription initiation regions from variousplant genes known to those of skill. Alternatively, the plant promotermay direct expression of nucleic acids of the invention in a specifictissue (tissue-specific promoters) or may be otherwise under moreprecise environmental control (inducible promoters). Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only in certain tissues, such as fruit,seeds or flowers.

Any of a number of promoters which direct transcription in plant cellscan be suitable. The promoter can be either constitutive or inducible.In addition to the promoters noted above, promoters of bacterial originthat operate in plants include the octopine synthase promoter, thenopaline synthase promoter and other promoters derived from native Tiplasmids. See, Herrara-Estrella et al. (1983) Nature 303:209. Viralpromoters include the 35S and 19S RNA promoters of cauliflower mosaicvirus. See, Odell et al. (1985) Nature 313:810. Other plant promotersinclude Kunitz trypsin inhibitor promoter (KTI), SCP1, SUP, UCD3, theribulose-1,3-bisphosphate carboxylase small subunit promoter and thephaseolin promoter. The promoter sequence from the E8 gene and othergenes may also be used. The isolation and sequence of the E8 promoter isdescribed in detail in Deikman & Fischer (1988) EMBO J 7:3315. Manyother promoters are in current use and can be coupled to an exogenousDNA sequence to direct expression.

If expression of a polypeptide from a cDNA is desired, a polyadenylationregion at the 3′-end of the coding region is typically included. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from, e.g., T-DNA.

A vector comprising sequences of the invention will typically include anucleic acid subsequence, a marker gene which confers a selectable, oralternatively, a screenable, phenotype on plant cells. For example, themarker can encode biocide tolerance, particularly antibiotic tolerance,such as tolerance to kanamycin, G418, bleomycin, hygromycin, orherbicide tolerance, such as tolerance to chlorosulforon, orphosphinothricin (the active ingredient in the herbicides bialaphos orBasta). See, e.g., Padgette et al. (1996) In: Herbicide-Resistant Crops(Duke, ed.), pp 53-84, CRC Lewis Publishers, Boca Raton (“Padgette,1996”). For example, crop selectivity to specific herbicides can beconferred by engineering genes into crops that encode appropriateherbicide metabolizing enzymes from other organisms, such as microbes.See, Vasil, (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91,CRC Lewis Publishers, Boca Raton) (“Vasil”, 1996).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

Transgenic or introgressed plants comprising nucleic acids of thepresent invention can be screened for transmission of the nucleic acidof the present invention by, for example, standard nucleic aciddetection methods or by immunoblot protocols.

A preferred embodiment of the invention is a transgenic plant that ishomozygous for the added heterologous nucleic acid; e.g., a transgenicplant that contains two added nucleic acid sequence copies. A homozygoustransgenic plant can be obtained by sexually mating (self-fertilizing) aheterozygous transgenic plant that contains a single added heterologousnucleic acid. Back-crossing to a parental plant and out-crossing with anon-transgenic plant can be used to introgress the heterologous nucleicacid into a selected background (e.g., an elite or exotic soybean line).

Methods for Charcoal Rot Drought Complex Tolerant Soybean Plants

Experienced plant breeders can recognize tolerant soybean plants in thefield, and can select the tolerant individuals or populations forbreeding purposes or for propagation. In this context, the plant breederrecognizes “tolerant” and “non-tolerant” or “susceptible”, soybeanplants.

Such plant breeding practitioners will appreciate that plant toleranceis a phenotypic spectrum consisting of extremes in tolerance,susceptibility and a continuum of intermediate tolerance phenotypes.Tolerance also varies due to environmental effects and the severity ofpathogen infection. Evaluation of phenotypes using reproducible assaysand tolerance scoring methods are of value to scientists who seek toidentify genetic loci that impart tolerance, conduct marker assistedselection for tolerant populations, and for introgression techniques tobreed a tolerance trait into an elite soybean line, for example.

In contrast to fortuitous field observations that classify plants aseither “tolerant” or “susceptible”, various systems are known forscoring the degree of plant tolerance or susceptibility. Thesetechniques can be applied to different fields at different times, andprovide approximate tolerance scores that can be used to characterize agiven strain regardless of growth conditions or location.

Ratings are assigned by evaluating all plants of a cultivar in a 2 rowby 15 foot plot. Cultivar scores are based on a 1 to 9 system where ascore of 9=no disease symptoms with normal plant growth; 8=very slightsymptoms including up to a 10% reduction in leaflet and overall canopysize with no wilting; 7=wilting beginning to appear at the uppermost twonodes; 6=wilting at the uppermost three nodes and leaflet yellowingbeginning appear; 5=Up to 5% plant death with wilting and yellowing ofleaflets occurring at the uppermost four nodes; 4=Up to 10% plant deathwith wilting and yellowing of leaflets occurring at the uppermost fournodes; 3=Up to 25% plant death with wilting and yellowing of leafletsoccurring at the uppermost four nodes; 2=up to 50% plant death;1=50-100% plant death. FIG. 8 gives a representative example ofcultivars with vastly different Charcoal Rot Drought Complex toleranceusing this scoring system.

Automated Detection/Correlation Systems of the Invention

In some embodiments, the present invention includes an automated systemfor detecting markers of the invention and/or correlating the markerswith a desired phenotype (e.g., tolerance). Thus, a typical system caninclude a set of marker probes or primers configured to detect at leastone favorable allele of one or more marker locus associated withtolerance or improved tolerance to Charcoal Rot Drought Complex. Theseprobes or primers are configured to detect the marker alleles noted inthe tables and examples herein, e.g., using any available alleledetection format, e.g., solid or liquid phase array based detection,microfluidic-based sample detection, etc.

For example, in one embodiment, the marker locus is Sct_028, Satt512,S60211-TB, Sat_117, P13158A, 563880-CB, S00415-1-A, S00705-1-A, andS02118-1-A, or any combination thereof, as well as any of the chromosomeintervals

-   -   (i) Satt286 and Satt371 (LG-C2);    -   (ii) Satt575 and Sat_136 (LG-E);    -   (iii) Satt467 and Satt416 (LG-B2);    -   (iv) Satt612 and A681_1 (LG-G);    -   (v) Sat_158 and A162_1 (LG-H);    -   (vi) Satt444 and Sat_331 (LG-B1);    -   (vii) Bng019_1 and Sct_191 (LG-C1);    -   (viii) A605_1 and A519_2 (LG-D1b); and,    -   (xi) Sat_306 and A363_3 (LG-N) or any combination thereof,        and the probe set is configured to detect the locus.

The typical system includes a detector that is configured to detect oneor more signal outputs from the set of marker probes or primers, oramplicon thereof, thereby identifying the presence or absence of theallele. A wide variety of signal detection apparatus are available,including photo multiplier tubes, spectrophotometers, CCD arrays, arraysand array scanners, scanning detectors, phototubes and photodiodes,microscope stations, galvo-scanns, microfluidic nucleic acidamplification detection appliances and the like. The preciseconfiguration of the detector will depend, in part, on the type of labelused to detect the marker allele, as well as the instrumentation that ismost conveniently obtained for the user. Detectors that detectfluorescence, phosphorescence, radioactivity, pH, charge, absorbance,luminescence, temperature, magnetism or the like can be used. Typicaldetector embodiments include light (e.g., fluorescence) detectors orradioactivity detectors. For example, detection of a light emission(e.g., a fluorescence emission) or other probe label is indicative ofthe presence or absence of a marker allele. Fluorescent detection isespecially preferred and is generally used for detection of amplifiednucleic acids (however, upstream and/or downstream operations can alsobe performed on amplicons, which can involve other detection methods).In general, the detector detects one or more label (e.g., light)emission from a probe label, which is indicative of the presence orabsence of a marker allele.

The detector(s) optionally monitors one or a plurality of signals froman amplification reaction. For example, the detector can monitor opticalsignals which correspond to “real time” amplification assay results.

System instructions that correlate the presence or absence of thefavorable allele with the predicted tolerance are also a feature of theinvention. For example, the instructions can include at least onelook-up table that includes a correlation between the presence orabsence of the favorable alleles and the predicted tolerance or improvedtolerance. The precise form of the instructions can vary depending onthe components of the system, e.g., they can be present as systemsoftware in one or more integrated unit of the system (e.g., amicroprocessor, computer or computer readable medium), or can be presentin one or more units (e.g., computers or computer readable media)operably coupled to the detector. As noted, in one typical embodiment,the system instructions include at least one look-up table that includesa correlation between the presence or absence of the favorable allelesand predicted tolerance or improved tolerance. The instructions alsotypically include instructions providing a user interface with thesystem, e.g., to permit a user to view results of a sample analysis andto input parameters into the system.

The system typically includes components for storing or transmittingcomputer readable data representing or designating the alleles detectedby the methods of the present invention, e.g., in an automated system.The computer readable media can include cache, main, and storage memoryand/or other electronic data storage components (hard drives, floppydrives, storage drives, etc.) for storage of computer code. Datarepresenting alleles detected by the method of the present invention canalso be electronically, optically, or magnetically transmitted in acomputer data signal embodied in a transmission medium over a networksuch as an intranet or internet or combinations thereof. The system canalso or alternatively transmit data via wireless, IR, or other availabletransmission alternatives.

During operation, the system typically comprises a sample that is to beanalyzed, such as a plant tissue, or material isolated from the tissuesuch as genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA,amplified RNA, or the like.

The phrase “allele detection/correlation system” in the context of thisinvention refers to a system in which data entering a computercorresponds to physical objects or processes external to the computer,e.g., a marker allele, and a process that, within a computer, causes aphysical transformation of the input signals to different outputsignals. In other words, the input data, e.g., amplification of aparticular marker allele is transformed to output data, e.g., theidentification of the allelic form of a chromosome segment. The processwithin the computer is a set of instructions, or “program”, by whichpositive amplification or hybridization signals are recognized by theintegrated system and attributed to individual samples as a genotype.Additional programs correlate the identity of individual samples withphenotypic values or marker alleles, e.g., statistical methods. Inaddition there are numerous e.g., C/C++ programs for computing, Delphiand/or Java programs for GUI interfaces, and productivity tools (e.g.,Microsoft Excel and/or SigmaPlot) for charting or creating look uptables of relevant allele-trait correlations. Other useful softwaretools in the context of the integrated systems of the invention includestatistical packages such as SAS, Genstat, Matlab, Mathematica, andS-Plus and genetic modeling packages such as QU-GENE. Furthermore,additional programming languages such as visual basic are also suitablyemployed in the integrated systems of the invention.

For example, tolerance marker allele values assigned to a population ofprogeny descending from crosses between elite lines are recorded in acomputer readable medium, thereby establishing a database correspondingtolerance alleles with unique identifiers for members of the populationof progeny. Any file or folder, whether custom-made or commerciallyavailable (e.g., from Oracle or Sybase) suitable for recording data in acomputer readable medium is acceptable as a database in the context ofthe present invention. Data regarding genotype for one or more molecularmarkers, e.g., ASH, SSR, RFLP, RAPD, AFLP, SNP, isozyme markers or othermarkers as described herein, are similarly recorded in a computeraccessible database. Optionally, marker data is obtained using anintegrated system that automates one or more aspects of the assay(s)used to determine marker(s) genotype. In such a system, input datacorresponding to genotypes for molecular markers are relayed from adetector, e.g., an array, a scanner, a CCD, or other detection devicedirectly to files in a computer readable medium accessible to thecentral processing unit. A set of system instructions (typicallyembodied in one or more programs) encoding the correlations betweentolerance and the alleles of the invention is then executed by thecomputational device to identify correlations between marker alleles andpredicted trait phenotypes.

Typically, the system also includes a user input device, such as akeyboard, a mouse, a touchscreen, or the like (for, e.g., selectingfiles, retrieving data, reviewing tables of maker information), and anoutput device (e.g., a monitor, a printer) for viewing or recovering theproduct of the statistical analysis.

Thus, in one aspect, the invention provides an integrated systemcomprising a computer or computer readable medium comprising a set offiles and/or a database with at least one data set that corresponds tothe marker alleles herein. The system also includes a user interfaceallowing a user to selectively view one or more of these databases. Inaddition, standard text manipulation software such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and database orspreadsheet software (e.g., spreadsheet software such as MicrosoftExcel™, Corel Quattro Pro™, or database programs such as MicrosoftAccess™ or Paradox™) can be used in conjunction with a user interface(e.g., a GUI in a standard operating system such as a Windows,Macintosh, Unix or Linux system) to manipulate strings of characterscorresponding to the alleles or other features of the database.

The systems optionally include components for sample manipulation, e.g.,incorporating robotic devices. For example, a robotic liquid controlarmature for transferring solutions (e.g., plant cell extracts) from asource to a destination, e.g., from a microtiter plate to an arraysubstrate, is optionally operably linked to the digital computer (or toan additional computer in the integrated system). An input device forentering data to the digital computer to control high throughput liquidtransfer by the robotic liquid control armature and, optionally, tocontrol transfer by the armature to the solid support is commonly afeature of the integrated system. Many such automated robotic fluidhandling systems are commercially available. For example, a variety ofautomated systems are available from Caliper Technologies (Hopkinton,Mass.), which utilize various Zymate systems, which typically include,e.g., robotics and fluid handling modules. Similarly, the common ORCA®robot, which is used in a variety of laboratory systems, e.g., formicrotiter tray manipulation, is also commercially available, e.g., fromBeckman Coulter, Inc. (Fullerton, Calif.). As an alternative toconventional robotics, microfluidic systems for performing fluidhandling and detection are now widely available, e.g., from CaliperTechnologies Corp. (Hopkinton, Mass.) and Agilent Technologies (PaloAlto, Calif.).

Systems for molecular marker analysis of the present invention can,thus, include a digital computer with one or more of high-throughputliquid control software, image analysis software for analyzing data frommarker labels, data interpretation software, a robotic liquid controlarmature for transferring solutions from a source to a destinationoperably linked to the digital computer, an input device (e.g., acomputer keyboard) for entering data to the digital computer to controlhigh throughput liquid transfer by the robotic liquid control armatureand, optionally, an image scanner for digitizing label signals fromlabeled probes hybridized, e.g., to markers on a solid support operablylinked to the digital computer. The image scanner interfaces with theimage analysis software to provide a measurement of, e.g., nucleic acidprobe label intensity upon hybridization to an arrayed sample nucleicacid population (e.g., comprising one or more markers), where the probelabel intensity measurement is interpreted by the data interpretationsoftware to show whether, and to what degree, the labeled probehybridizes to a marker nucleic acid (e.g., an amplified marker allele).The data so derived is then correlated with sample identity, todetermine the identity of a plant with a particular genotype(s) forparticular markers or alleles, e.g., to facilitate marker assistedselection of soybean plants with favorable allelic forms of chromosomesegments involved in agronomic performance (e.g., tolerance or improvedtolerance).

Optical images, e.g., hybridization patterns viewed (and, optionally,recorded) by a camera or other recording device (e.g., a photodiode anddata storage device) are optionally further processed in any of theembodiments herein, e.g., by digitizing the image and/or storing andanalyzing the image on a computer. A variety of commercially availableperipheral equipment and software is available for digitizing, storingand analyzing a digitized video or digitized optical image, e.g., usingPC (Intel x86 or Pentium chip-compatible DOS™, OS2™ WINDOWS™, WINDOWSNT™ or WINDOWS 95™ based machines), MACINTOSH™, LINUX, or UNIX based(e.g., SUN™ work station) computers.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only, and persons skilledin the art will recognize various reagents or parameters that can bealtered without departing from the spirit of the invention or the scopeof the appended claims.

Example 1

Intergroup Allele Frequency Distribution Analysis

Two independent allele frequency distribution analyses were undertakento identify soybean genetic marker loci associated with tolerance toCRDC. By identifying such genetic markers, marker assisted selection(MAS) can be used to improve the efficiency of breeding for improvedtolerance of soybean to CRDC.

Soybean Lines and Tolerance Scoring

The plant varieties used in the analysis were from diverse sources,including elite germplasm, commercially released cultivars and otherpublic lines representing a broad range of germplasm. The lines used inthe study had a broad maturity range varying from group 2 to group 4.

Two groups of soybean lines were assembled for each analysis based ontheir phenotypic extremes in tolerance to CRDC, where the plants weresorted into either highly susceptible or highly tolerant varieties. Theclassifications of tolerant and susceptible were based solely onobservations of fortuitous, naturally occurring disease incidence infield tests over several years and greenhouse observations. The degreeof plant tolerance to Charcoal Rot infection varied widely, as measuredusing a scale from one (highly susceptible) to nine (highly tolerant).Generally, a score of two (2) or three (3) indicated the mostsusceptible strains, and a score of seven (7) or eight (8) was assignedto the most tolerant lines. A score of one (1) was generally not used,as soybean strains with such extremely high susceptibility were nottypically propagated. Tolerance scores of nine (9) were reserved fortolerance levels that are very rare and generally not observed inexisting germplasm. If no disease was present in a field, no tolerancescoring was done. However, if a disease did occur in a specific fieldlocation, all of the lines in that location were scored. Scores for teststrains accumulated over multiple locations and multiple years, and anaveraged (e.g., consensus) score was ultimately assigned to each line.

Individual fields showing Charcoal Rot were monitored for diseasesymptoms during the vegetative stages but typically appear in the earlyto late reproductive stages. Data collection was typically done in threeor four successive scorings about seven days apart. Scorings continueduntil worsening symptoms can no longer be quantified or until thesymptoms are confounded by other factors such as other diseases, insectpressure, severe weather, or advancing maturity.

In assessing association of markers to tolerance, a qualitative“intergroup allele frequency distribution” comparison approach was used.Using this approach, those soybean lines that were considered to berepresentative of either the tolerant or susceptible classes were usedfor assessing association. A list of tolerant lines was constructed,where strains having a tolerance score of 7 or greater were considered“tolerant.” Similarly, soybean lines with scores of three or less werecollectively considered susceptible. Only lines that could be reliablyplaced into the two groups were used. Once a line is included in the“tolerant” or “susceptible” group, it was treated as an equal in thatgroup, i.e., the actual quantitative ratings were not used.

In the study, 29 soybean lines were identified that were consideredtolerant in the phenotypic spectrum; these plants formed the “TOLERANT”group. Also, 38 soybean lines were identified that were judged to besusceptible to Charcoal Rot; these strains formed the “SUSCEPTIBLE”group.

Soybean Genotyping

Each of the tolerant and susceptible lines was genotyped with SSR andSNP markers that span the soybean genome using techniques well known inthe art. The genotyping protocol consisted of collecting young leaftissue from eight individuals from each tolerant and resistant soybeanstrain, pooling (i.e., bulking) the leaf tissue from the eightindividuals, and isolating genomic DNA from the pooled tissue. Thesoybean genomic DNA was extracted by the CTAB method, as described inMaroof et al. (1984) Proc Natl Acad Sci USA 81:8014-8018.

The isolated genomic DNA was then used in PCR reactions usingamplification primers specific for a large number of markers thatcovered all chromosomes in the soybean genome. The length of the PCRamplicon or amplicons from each PCR reaction were characterized. Thelength of the amplicons generated in the PCR reactions were compared toknown allele definitions for the various markers (see, e.g., FIG. 4),and allele designations were assigned. SNP-type markers were genotypedusing an ASH protocol.

Intergroup Allele Frequency Analysis

An “Intergroup Allele Frequency Distribution” analysis was conductedusing GeneFlow™ version 7.0 software. An intergroup allele frequencydistribution analysis provides a method for finding non-randomdistributions of alleles between two phenotypic groups.

During processing, a contingency table of allele frequencies isconstructed and from this a G-statistic and probability are calculated(the G statistic is adjusted by using the William's correction factor).The probability value is adjusted to take into account the fact thatmultiple tests are being done (thus, there is some expected rate offalse positives). The adjusted probability is proportional to theprobability that the observed allele distribution differences betweenthe two classes would occur by chance alone. The lower that probabilityvalue, the greater the likelihood that the Charcoal Rot infectionphenotype and the marker will co-segregate. A more complete discussionof the derivation of the probability values can be found in theGeneFlow™ version 7.0 software documentation. See, also, Sokal & Rolf,(1981), Biometry: The Principles and Practices of Statistics inBiological Research, 2nd ed., San Francisco, W. H. Freeman and Co.

The underlying logic is that markers with significantly different alleledistributions between the tolerant and susceptible groups (i.e., nonrandom distributions) might be associated with the trait and can be usedto separate them for purposes of marker assisted selection of soybeanlines with previously uncharacterized tolerance or susceptibility toCharcoal Rot. The present analysis examined one marker locus at a timeand determined if the allele distribution within the tolerant group issignificantly different from the allele distribution within thesusceptible group. A statistically different allele distribution is anindication that the marker is linked to a locus that is associated withreaction to Charcoal Rot. In this analysis, unadjusted probabilitiesless than one are considered significant (the marker and the phenotypeshow linkage disequilibrium), and adjusted probabilities less thanapproximately 0.05 are considered highly significant. Allele classesrepresented by less than 5 observations across both groups were notincluded in the statistical analysis. In this analysis, 444 marker locihad enough observations for analysis.

This analysis compares the plants' phenotypic score with the genotypesat the various loci. This type of intergroup analysis neither generatesnor requires any map data. Subsequently, map data (for example, acomposite soybean genetic map) is relevant in that multiple significantmarkers that are also genetically linked can be considered ascollaborating evidence that a given chromosomal region is associatedwith the trait of interest.

Results

FIG. 1 provides a table listing the soybean markers that demonstratedlinkage disequilibrium with the Charcoal Rot tolerance/susceptibilityphenotype. Also indicated in that figure are the chromosomes on whichthe markers are located and their approximate map position relative toother known markers, given in cM, with position zero being the first(most distal) marker known at the beginning of the chromosome. These mappositions are not absolute, and represent an estimate of map position.The statistical probabilities that the marker allele and tolerancephenotype are segregating independently are reflected in the adjustedprobability values.

FIG. 2 provides the PCR primer sequences that were used to genotypethese marker loci. FIG. 2 also provides the pigtail sequence used on the5′ end of the right SSR-marker primers and the number of nucleotides inthe repeating element in the SSR. The observed alleles that are known tooccur for these marker loci are provided in the allele dictionary inFIG. 4.

Out of 444 loci studied, simple sequence repeat (SSR) or singlenucleotide polymorphism (SNP) loci having adjusted probability valuesfor independent assortment with Charcoal Rot tolerance of less thanapproximately 0.05 were identified (see, FIG. 1). The statisticalprobabilities that the marker allele and tolerance phenotype aresegregating independently are reflected in the Adjusted Probabilityvalues.

Discuission

There are a number of ways to use the information provided in thisanalysis for the development of improved soybean varieties. Oneapplication is to use the associated markers (or more based on a higherprobability cutoff value) as candidates for mapping QTL in specificpopulations that are segregating for plants having tolerance to CharcoalRot infection. In this application, one proceeds with conventional QTLmapping in a segregating population, but focusing on the markers thatare associated with Charcoal Rot infection tolerance, instead of usingmarkers that span the entire genome. This makes mapping efforts morecost-effective by dramatically reducing lab resources committed to theproject. For example, instead of screening segregating populations witha large set of markers that spans the entire genome, one would screenwith only those few markers that met some statistical cutoff in theintergroup allele association study. This will not only reduce the costof mapping but will also eliminate false leads that will undoubtedlyoccur with a large set of markers. In any given cross, it is likely thatonly a small subset of the associated markers will actually becorrelated with tolerance to Charcoal Rot infection. Once the fewrelevant markers are identified in any tolerant parent, future markerassisted selection (MAS) efforts can focus on only those markers thatare important for that source of tolerance. By pre-selecting lines thathave the allele associated with tolerance via MAS, one can eliminate theundesirable susceptible lines and concentrate the expensive fieldtesting resources on lines that have a higher probability of beingtolerant to Charcoal Rot infection.

Example 2

Trait Allele Correlation Analysis

One trait allele correlation analysis was conducted using GeneFlow v.7.0 to identify soybean genetic marker loci associated with tolerance toCRDC. By identifying such genetic markers, marker assisted selection(MAS) can be used to improve the efficiency of breeding for improvedtolerance of soybean to CRDC.

Soybean Lines and Tolerance Scoring

One hundred and sixty seven lines were characterized for their CharcoalRot Drought Tolerance score. The plant varieties used in the analysiswere from diverse sources, including elite germplasm, commerciallyreleased cultivars and other public lines representing a broad range ofgermplasm. The lines used in the study had a broad maturity rangevarying from group 2 to group 4.

The classifications of the lines for CRDC reaction were in a continuousrange from 1 (susceptible) up to 8 (highly tolerant) and scores werebased solely on observations of fortuitous, naturally occurring diseaseincidence in field tests over several years and greenhouse observations.Generally, a score of two (2) or three (3) indicated the mostsusceptible strains, and a score of seven (7) or eight (8) was assignedto the most tolerant lines. Tolerance scores of nine (9) were reservedfor tolerance levels that are very rare and generally not observed inexisting germplasm. If no disease was present in a field, no tolerancescoring was done. However, if a disease did occur in a specific fieldlocation, all of the lines in that location were scored. Scores for teststrains accumulated over multiple locations and multiple years, and anaveraged (e.g., consensus) score was ultimately assigned to each line.

Individual fields showing Charcoal Rot were monitored for diseasesymptoms during the vegetative stages but typically appear in the earlyto late reproductive stages. Data collection was typically done in threeor four successive scorings about seven days apart. Scorings continueduntil worsening symptoms can no longer be quantified or until thesymptoms are confounded by other factors such as other diseases, insectpressure, severe weather, or advancing maturity.

Soybean Genotyping

Each of the tolerant and susceptible lines was genotyped with SSR andSNP markers that span the soybean genome using techniques well known inthe art. The genotyping protocol consisted of collecting young leaftissue from eight individuals from each tolerant and resistant soybeanstrain, pooling (i.e., bulking) the leaf tissue from the eightindividuals, and isolating genomic DNA from the pooled tissue. Thesoybean genomic DNA was extracted by the CTAB method, as described inMaroof et al. (1984) Proc Natl Acad Sci USA 81:8014-8018.

The isolated genomic DNA was then used in PCR reactions usingamplification primers specific for a large number of markers thatcovered all chromosomes in the soybean genome. The length of the PCRamplicon or amplicons from each PCR reaction were characterized. Thelength of the amplicons generated in the PCR reactions were compared toknown allele definitions for the various markers (see, e.g., FIG. 4),and allele designations were assigned. SNP-type markers were genotypedusing an ASH protocol.

Trait Allele Correlation Analysis

For the Trait Allele Correlation report you must select accessions,markers and a single trait. For each allele at each selected marker, thereport will show you the effect of having 0, 1 or 2 doses of that alleleon the trait of interest. For each dosage comparison it calculates at-statistic, probability and adjusted probability by comparing the meansof two different dosage classes. The adjusted probability gives you abetter idea of the experiment-wise significance given the number ofalleles being tested, and is calculated as P_adj=(1−((1−Prob)**n)) wheren is the number of tests being done in this analysis (see, ExperimentalDesign: Procedures for the Behavioral Sciences). A more completediscussion of the derivation of the probability values can be found inthe GeneFlow version 7.0 software documentation. See also, Sokal & Rolf,(1995) Biometry 3rd ed., San Francisco, W.H. Freeman and Co.

Results

FIG. 1 provides a table listing the soybean markers that demonstratedlinkage disequilibrium with the CRDC trait scores of 167 lines. Alsoindicated in that figure are the chromosomes on which the markers arelocated and their approximate map position relative to other knownmarkers, given in cM, with position zero being the first (most distal)marker known at the beginning of the chromosome. These map positions arenot absolute, and represent an estimate of map position. The statisticalprobabilities that the marker allele and tolerance phenotype aresegregating independently are reflected in the adjusted probabilityvalues.

FIG. 2 provides the PCR primer sequences that were used to genotypethese marker loci. FIG. 2 also provides the pigtail sequence used on the5′ end of the right SSR-marker primers and the number of nucleotides inthe repeating element in the SSR. The observed alleles that are known tooccur for these marker loci are provided in the allele dictionary inFIG. 4.

Out of 444 loci studied, simple sequence repeat (SSR) or singlenucleotide polymorphism (SNP) loci having adjusted probability valuesfor independent assortment with Charcoal Rot tolerance of less thanapproximately 0.05 were identified (see, FIG. 1). The statisticalprobabilities that the marker allele and tolerance phenotype aresegregating independently are reflected in the Adjusted Probabilityvalues.

Discussion

There are a number of ways to use the information provided in thisanalysis for the development of improved soybean varieties. Oneapplication is to use the associated markers (or more based on a higherprobability cutoff value) as candidates for mapping QTL in specificpopulations that are segregating for plants having tolerance to CharcoalRot infection. In this application, one proceeds with conventional QTLmapping in a segregating population, but focusing on the markers thatare associated with Charcoal Rot infection tolerance, instead of usingmarkers that span the entire genome. This makes mapping efforts morecost-effective by dramatically reducing lab resources committed to theproject. For example, instead of screening segregating populations witha large set of markers that spans the entire genome, one would screenwith only those few markers that met some statistical cutoff in theintergroup allele association study. This will not only reduce the costof mapping but will also eliminate false leads that will undoubtedlyoccur with a large set of markers. In any given cross, it is likely thatonly a small subset of the associated markers will actually becorrelated with tolerance to Charcoal Rot infection. Once the fewrelevant markers are identified in any tolerant parent, future markerassisted selection (MAS) efforts can focus on only those markers thatare important for that source of tolerance. By pre-selecting lines thathave the allele associated with tolerance via MAS, one can eliminate theundesirable susceptible lines and concentrate the expensive fieldtesting resources on lines that have a higher probability of beingtolerant to Charcoal Rot infection.

Example 3

Charcoal Rot Drought Complex Tolerance Phenotypic Assay

A field nursery was established in a region of Southwestern Missourithat was known for severe Charcoal rot symptoms caused by the fungusMacrophomina phaseolina. Management practices that promote severeCharcoal Rot Drought Complex symptoms were followed including: earlyplanting date, high seeding rate, reduced tillage, and low soilfertility. Genotypes were blocked together by similar maturity andreplicated three times. Each genotype was grown in a two row plotmeasuring 5 ft. wide×15 ft. long. Ratings were taken during theseed-filling stages when the plant's demand for water is the greatest.The first rating was taken during the R4-R5 stage and the final ratingwas taken during the R5-R6 growth stage.

Cultivar scores are based on a 1 to 9 system where a score of 9=nodisease symptoms with normal plant growth; 8=very slight symptomsincluding up to a 10% reduction in leaflet and overall canopy size withno wilting; 7=wilting beginning to appear at the uppermost two nodes;6=wilting at the uppermost three nodes and leaflet yellowing beginningappear; 5=Up to 5% plant death with wilting and yellowing of leafletsoccurring at the uppermost four nodes; 4=Up to 10% plant death withwilting and yellowing of leaflets occurring at the uppermost four nodes;3=Up to 25% plant death with wilting and yellowing of leaflets occurringat the uppermost four nodes; 2=up to 50% plant death; 1=50-100% plantdeath. FIG. 8 gives a representative example of cultivars with vastlydifferent Charcoal Rot Drought Complex tolerance using this scoringsystem.

Example 4

Genotyping the Mapping Population

For genotypic data, DNA was isolated from the collected leaves from 368progeny. Leaf tissue was punched and the tissue was genotyped using SSRmarkers. A total of 333 SNP-based markers were screened against themapping population to identify polymorphic markers potentiallyassociated with the CRDC phenotype.

MapManager-QTX was used for both genetic mapping and QTL analysis. The2000 permutation tests were used to establish the threshold forstatistical significance (LOD ratio statistic—LRS). The mean score wereused for QTL mapping. The LRS threshold at P=0.05 is 9.1 and at P=0.01is 17.9.

The 333 SNP-based markers were screened for the population. The 333 SNPmarkers coalesced into 32 linkage groups, with 6 markers being unlinked.The number of markers for each linkage group ranged from 2 to 27.

One major QTL was identified on linkage group G (Table 1) with theclosely linked marker of S01954-1-A. Several public markers in thisregion, Satt472, Satt191, Sat_117, and Sct_187, all are recognized to beassociated with CRDC tolerance. This QTL has an LRS score of 32.5 andexplains, on average, approximately 10% of the observed phenotypicvariation.

TABLE 1 Interval mapping output for linkage group G Marker Map Stat %Add S01954-1-A 0.16 33.8 10 0.29 0.17 34.9 10 0.30 0.18 29.2 9 0.26

One major QTL was identified on linkage group C1 (Table 2) with theclosely linked marker of S00415-1-A. Several public markers in thisregion, Satt607, Satt190, Satt139, Satt136, Sat_416, and Sat_085, allare recognized to be associated with CRDC tolerance. This QTL has an LRSscore of 35.4 and explains, on average, approximately 10% of theobserved phenotypic variation.

TABLE 2 Interval mapping output for linkage group C1 Marker Map Stat %Add S00415-1-A 0.02 37.0 10 0.29 0.03 36.8 10 0.29 0.04 34.0 10 0.28

An interaction analysis was run on the loci from linkage group G andlinkage group C1. No evidence of direct epistatic interaction between Gand C1 was found.

One minor QTL was identified on linkage group D1b (Table 3) with theclosely linked marker of S00705-1-A. Several public markers in thisregion, Satt428, Sat_169, Satt644, Satt041, and Satt546, all arerecognized to be associated with CRDC tolerance. This QTL has an LRSscore of 11.5 and explains, on average, approximately 4% of thevariation.

TABLE 3 Interval mapping output for linkage group D1b Marker Map Stat %Add S00705-1-A 0.11 11.5 3 0.16 0.12 12.3 4 0.17 0.13 12.6 4 0.18

One minor QTL was identified on linkage group N (Table 4) with theclosely linked marker of S02118-1-A. Several public markers in thisregion, Satt022, Sat_125, A363_3 all are recognized to be associatedwith CRDC tolerance. This QTL has an LRS score of 9.4 and explains, onaverage, approximately 3% of the variation.

TABLE 4 Interval mapping output for linkage group N Marker Map Stat %Add S02118-1-A 0.01 7.0 2 0.13 0.02 8.5 2 0.14 0.02 10.1 3 0.16

There were notable environmental difference between 2005 and 2006. The2006 environment had much greater drought conditions versus 2005,resulting in added charcoal rot and physiological stress on the plant.The environments did effect the phenotypic distribution of thepopulation. The 2005 environment resulted in a much broader charcoal rotphenotypic distribution, with the parents having a much greaterphenotypic separation compared to the 2006 environment as show in thestatistics below:

2005 Statistics 2006 Statistics Mean 6.408108 4.601333 Standard Error0.053813 0.051677 Median 6.5 4.666667 Mode 7 4.333333 Standard Deviation1.035123 1.000719 Sample Variance 1.071479 1.001439 Kurtosis 0.524948−0.09353 Skewness −0.55391 −0.19395 Range 6.5 5 Minimum 2 2 Maximum 8.57

FIG. 1 provides a table listing the soybean markers that demonstratedlinkage disequilibrium with the CRDC trait scores from pools orpopulations as noted. Also indicated in that figure are the chromosomeson which the markers are located and their approximate map positionrelative to other known markers, given in cM, with position zero beingthe first (most distal) marker known at the beginning of the chromosome.These map positions are not absolute, and represent an estimate of mapposition. The statistical probabilities that the marker allele andtolerance phenotype are segregating independently are reflected in theadjusted probability values.

What is claimed is:
 1. A method of producing a soybean plant orgermplasm that displays tolerance or improved tolerance to Charcoal RotDrought Complex (CRDC), the method comprising: (a) isolating nucleicacids from a genome of a first soybean plant or germplasm; (b) detectingin the first soybean plant or germplasm at least one allele of aquantitative trait locus that is associated with the tolerance orimproved tolerance, wherein said quantitative trait locus is localizedto a chromosomal interval flanked by and including markers Satt575 andSat_136 on linkage group E, wherein the at least one allele is detectedusing a composition comprising a detectable label; (c) selecting saidfirst soybean plant or germplasm, or selecting a progeny of said firstsoybean plant or germplasm, wherein the plant, germplasm or progenythereof comprise at least one allele associated with tolerance orimproved tolerance to CRDC; and (d) crossing said selected first soybeanplant or germplasm with a second soybean plant or germplasm tointrogress said quantitative trait locus into progeny soybean germplasm.2. The method of claim 1, wherein said quantitative trait locus isfurther localized to a chromosomal interval flanked by and includingSatt411 and Satt720 on linkage group E.
 3. The method of claim 1,wherein said quantitative trait locus is Satt512.
 4. The method of claim1, wherein the at least one allele of the quantitative trait locuscomprises Satt512:allele-2 or Satt512:allele-5.
 5. The method of claim1, wherein the second soybean plant or germplasm displays less toleranceto CRDC as compared to the first soybean plant or germplasm, and whereinthe introgressed soybean plant or germplasm displays an increasedtolerance to CRDC as compared to the second plant or germplasm.
 6. Themethod of claim 1, further comprising a) analyzing progeny soybeangermplasm to determine the presence of tolerance to CRDC; and b)selecting progeny soybean germplasm that tests positive for the presenceof tolerance to CRDC as being soybean germplasm into which germplasmhaving said quantitative trait locus has been introgressed.